Compositions and methods for neuralgenesis

ABSTRACT

The present invention relates to novel compositions and methods to produce 3D organ equivalents of the brain (i.e. “mini-brains”). The invention also relates to methods of using human induced pluripotent stem cells, a combination of growth and other soluble factors and gyratory shaking. Cells from healthy or diseased donors or animals can be used to allow testing different genetic backgrounds. The model can be further enhanced by using genetically modified cells, adding micro-glia or their precursors or indicator cells (e.g. with reporter genes or tracers) as well as adding endothelial cells to form a blood-brain-barrier.

RELATED APPLICATIONS

This application is a continuation application, filed under 35 U.S.C. §120, of U.S. application Ser. No. 16/077,411, filed on Aug. 10, 2018,which is a national stage application, filed under 35 U.S.C. § 371, ofInternational Stage Application No. PCT/US2017/017464, filed on Feb. 10,2017, which claims the benefit of priority under 35 U.S.C. § 119(e) toU.S. Provisional Application No. 62/294,112, filed Feb. 11, 2016, all ofwhich are incorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The invention was made with government support under the following grantawarded by the National Institute of Health (NIH): U18TR000547. Thegovernment has certain rights in the invention.

BACKGROUND

Simple neural in vitro systems do not reflect the physiology, cellularinteractions, or genetics of mammalian brain tissue. Accordingly, thereis an unmet need to develop human models of brain disorders and/ordiseases.

SUMMARY

Aspects of the invention are directed to an in vitro brainmicrophysiological system (BMPS). In embodiments, the BMPS comprises aleast one neural cell type aggregated into a spheroid mass and apopulation of microglia-like cells. The in vitro BMPS can beelectrophysiologically active in a spontaneous manner.

In one embodiment, the micro-glia like cells comprise microglia,microglia precursor cells, or a combination thereof. The micro-glia likecells can comprise monocytes, human monocytes, pro-monocyte cell lines,hematopoietic stem cells, isolated microglia, immortalized microglia, orcombinations thereof. In embodiments, the monocytes comprise adultcell-derived monocytes, embryonic cell-derived monocytes, or acombination thereof. The monocytes can comprise embryonic stem cell(ESC)-derived monocytes, induced pluripotent stem cell (iPSC)-derivedmonocytes, or a combination thereof. In certain embodiments, theisolated microglia comprise adult microglia, fetal microglia, or acombination thereof. The microglia-like cells can be derived fromsomatic cells, neuronal cells, myeloid progenitor cells, or acombination thereof. In embodiments, the microglia-like cells expressone or more of the following biomarkers: HLA-DR, Iba1, CD14, CX3CR1,F4/80, CD80, CD86, CD36, iNOS, COX2, ARG1, PPARy, SOCS-3, TMEM119,Mertk, Ax1, CD11b, CD11c, P2RY12, CD45, CD68, CD40, B7, ICAM-1 or anycombination thereof.

In some embodiments, the BMPS expresses receptors associated withmicroglia function. In embodiments, the receptors associated withmicroglia function comprise CCL2, CX3CL, RAGE, NLRP3, SR-AI, TREM2,FPRL1/FPR2, CD36, CD33, C5a, CR1, CR3/Mac-1, FcRs, FPRs, TLRs, or acombination thereof.

In embodiments, the BMPS is configured to elicit a pro-inflammatoryresponse, an anti-inflammatory response, or a combination thereof. TheBMPS can be configured to elicit a pro-inflammatory response to viralinfection, LPS exposure, or a combination thereof. The BMPS beingconfigured to elicit an anti-inflammatory response to IL-3, IL-4, IL-10,IL-13, IL-1β, IL-6, TNF-α, TGF-β, or a combination thereof.

In certain embodiments, the microglia-like cells comprise about 20% orless of the BMPS.

In embodiments, the at least one neuronal cell type comprises a matureneuron, a glial cell, or a combination thereof. The at least one neuralcell type can further comprise astrocytes, polydendrocytes,oligodendrocytes, or combinations thereof.

In various exemplary embodiments, the in vitro BMPS has neuralcharacteristics selected from the group consisting of synaptogenesis,neuron-neuron interactions, neuronal-glial interactions, axonmyelination, cell migration, neurological development, diseasephenotypes, or combinations thereof. Exemplary disease processphenotypes include autophagy, Integrated stress response, non-sensemediated decay, lesions, amyloid deposition, plaque formation, proteinaggregation, or combinations thereof.

In embodiments, the at least one neural cell type express one or morebiomarker selected from the group consisting of MBP, PLP, NG2, Olig1,Olig2, Olig 3, OSP, MOG, SOX10, neurofilament 200 (NF200), GRIN1, GAD1,GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, TH, TUBIII, NEUN, SLC1A6, andany combination thereof. The at least one neuronal cell type cancomprise one or more genetically modified cells. In embodiments, the oneor more genetically modified cells comprise one or more reporter genes.

In certain embodiments, the spheroid mass comprises a diameter that isabout 1000 μm or less. The spheroid mass can comprise a diameter that isabout 500 μm or less.

In embodiments, the BMPS comprises one or more endothelial cells,pericytes, or a combination thereof capable of forming ablood-brain-barrier.

Other aspects are directed to a method of reproducibly producing an invitro brain microphysiological system (BMPS). In embodiments, the methodcomprises any one or more of the following steps: inducing one or morepluripotent stem cell (PSC) types; differentiating the one or more PSCtypes to form one or more neural progenitor cell (NPC) types; exposingthe one or more NPC types to gyratory shaking or stirring;differentiating the one or more NPC types into one or more neural celltypes aggregated into a spheroid mass; and adding microglia-like cells.In embodiments, the micro-glia like cells comprise mature microglia,monocytes, human monocytes, pro-monocyte cell lines, PSC-derivedmonocytes, hematopoetic stem cells, isolated microglia, immortalizedmicroglia, or combinations thereof. In embodiments, the one or morepluripotent stem cells are selected from the group consisting of humanor animal embryonic stem cells, iPSC, adult stem cells, fibroblasts,embryonic fibrobflasts, peripheral blood mononuclear cells, neuronalprecursor cells, mesenchymal stem cells, neuronal cells, glial cells,and combinations thereof. The microglia-like cells can be added duringan early stage (before BMPS differentiation) or late stage (after BMPSdifferentiation). In alternate embodiments, the microglia are generatedin parallel with the BMPS. In certain embodiments, gyratory shakingcomprises constant or regular gyratory shaking or stirring for 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 ormore weeks.

The present invention provides brain microphysiological systems (BMPS)that can be produced from induced pluripotent stem cells (iPSCs).Furthermore, the invention provides for reproducible BMPS thatdifferentiate into mature neurons and glial cells (astrocytes andoligodendrocytes) in the central nervous system. This model iselectrophysiologically active in a spontaneous manner and may bereproduced with patient cells. The derivation of 3D BMPS from iPSCs hasapplications in the study and treatment of neurological diseases.

In an aspect, the disclosure provides an in vitro brainmicrophysiological system (BMPS), comprising two or more neural celltypes aggregated into a spheroid mass, wherein the spheroid mass has adiameter that is less than about 500 μm and the in vitro BMPS iselectrophysiologically active in a spontaneous manner.

In an embodiment, the two or more neural cell types comprise at least amature neuron and glial cell.

In an embodiment, the two or more neural cell types further comprisecells selected from the group consisting of astrocytes, polydendrocytes,oligodendrocytes, and combinations thereof.

In an embodiment, the in vitro BMPS has neural characteristics selectedfrom the group consisting of synaptogenesis, neuron-neuron interactions,neuronal-glial interactions, axon myelination, and combinations thereof.

In an embodiment, two or more neural cell types of the in vitro BMPSexpress one or more biomarker selected from the group consisting ofGRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII,NEUN, SLC1A6, and any combination thereof.

In an aspect, the disclosure provides a synthetic neurological organcomprising two or more neural cell types aggregated into a spheroidmass, wherein the spheroid mass has a diameter that is less than 500 μmand the in vitro BMPS is electrophysiologically active in a spontaneousmanner. In an embodiment, the two or more neural cell types comprise atleast a mature neuron and glial cells.

In an embodiment, the mature neuron and glial cells further comprisecells selected from the group consisting of astrocytes, polydendrocytes,oligodendrocytes, and combinations thereof.

In an embodiment, the synthetic neurological organ further comprisesneural characteristics selected from the group consisting ofsynaptogenesis, neuron-neuron interactions, neuronal-glial interactions,axon myelination, and combinations thereof.

In an embodiment, the synthetic neurological organ mimics themicroenvironment of the central nervous system (CNS).

In an aspect, the disclosure provides a method of reproducibly producingan in vitro brain microphysiological system (BMPS), comprising: inducingone or more pluripotent stem cell (PSC) types; differentiating the oneor more PSC types to form one or more neural progenitor cell (NPC)types; exposing the one or more NPC types to gyratory shaking orstirring; and differentiating the one or more NPC types into one or moreneural cell types aggregated into a spheroid mass, wherein the spheroidmass has a diameter that is less than 500 μm.

In an embodiment, the one or more pluripotent stem cells are selectedfrom the group consisting of human or animal embryonic stem cells, iPSC,adult stem cells, fibroblasts, embryonic fibroblasts, peripheral bloodmononuclear cells, neuronal precursor cells, mesenchymal stem cells, andcombinations thereof.

In an embodiment, inducing further comprises: adding micro-glia ormicro-glia precursor cells.

In an embodiment, the micro-glia or micro-glia precursor cells areselected from the group consisting of monocytes, human monocytes,pro-monocyte cell lines, iPSC-derived monocytes, hematopoetic stemcells, isolated microglia, immortalized microglia, and combinationsthereof.

In an embodiment, gyratory shaking comprises constant or regulargyratory shaking or stirring for 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, or 8 or more weeks.

In an embodiment, the one or more growth factors are selected from thegroup consisting of GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF,CNTF, and any combination thereof.

In an aspect, the disclosure provides a method of cryopreserving an invitro brain microphysiological system (BMPS), comprising:differentiating BMPS aggregates into one or more mature neurons;incubating the aggregates in a cryopreserving medium; and exposing theaggregates to freezing temperatures of −60° C. or colder.

In an embodiment, differentiating further comprises: inducingdifferentiation of one or more pluripotent stem cell types by incubationwith one or more growth factors.

In an embodiment, the one or more pluripotent stem cells are selectedfrom a group consisting of human or animal embryonic stem cells, iPSC,adult stem cells, fibroblasts, embryonic fibroblasts, peripheral bloodmononuclear cells, neuronal precursor cells, mesenchymal stem cells, andcombinations thereof.

In an embodiment, inducing further comprises: adding micro-gliaprecursor cells.

In an embodiment, micro-glia precursor cells are selected from the groupconsisting of monocytes, human monocytes, iPSC-derived monocytes,hematopoetic stem cells, pro-monocyte cell lines, isolated microglia,immortalized microglia, and combinations thereof.

In an embodiment, the one or more growth factors are selected from thegroup consisting of GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF,CNTF, and any combination thereof.

In an embodiment, the cryopreserving medium is a medium selected fromthe group consisting of regular cryopreservation medium (95% FBS and 5%DMSO), STEMdiff Neural Progenitor Freezing Medium (Stem CellsTechnologies), solutions with cryoprotectants, and combinations thereof.

In an embodiment, exposing the aggregates to freezing temperaturesfurther comprises freezing aggregates over a temperature gradient ofabout 1° C. per hour to below −60° C. over up to 48 hours.

In an embodiment, cryopreserving further comprises additives selectedfrom the group consisting of DMSO, HES, glycerol, serum, and anycombination or derivative thereof.

In an aspect, the disclosure provides a method of transporting a brainmicrophysiological system (BMPS) or mini-brain, comprising: producingthe BMPS or mini-brain of claim 1, incubating the BMPS or mini-brain at37° C., and maintaining the temperature at 37° C. with constantapplication of heat while moving the BMPS or mini-brain.

In an embodiment, maintaining the temperature comprises use of heatingpads, heaters, insulation, insulated boxes, heat packs, electricblankets, chemical pads, and combinations thereof.

In an aspect, the disclosure provides a method of studying aneurological disease or disorder comprising: producing an in vitro brainmicrophysiological system (BMPS); exposing the in vitro BMPS toconditions that replicate or induce the neurological disease ordisorder; adding an agent to treat the neurological disease or disorder;and assessing the effect of the agent on the neurological disease ordisorder.

In an embodiment, the neurological disease or disorder is selected fromthe group consisting of neurodegenerative disorder, muscular dystrophy,Parkinson's Disease, Huntington's Disease, Autism Spectrum Disorder andother neurodevelopmental disorders, Down's Syndrome, Multiple Sclerosis,Amyotrophic lateral sclerosis, brain cancer, encephalitis, infection,trauma, stroke, and paralysis.

In an aspect, the disclosure provides a method of treating a patienthaving a neurological disease or disorder, comprising: extracting a stemcell from the patient with a genetic background pre-disposed for theneurological disease or disorder; producing a brain microphysiologicalsystem (BMPS) or mini-brain with the genetic background; treating theBMPS or mini-brain with an agent targeting the neurological disease ordisorder; and assessing the effect of the agent on the BMPS ormini-brain.

In an embodiment, the neurological disease or disorder is selected fromthe group consisting of neurodegenerative disorder, muscular dystrophy,Parkinson's Disease, Huntington's Disease, Autism Spectrum Disorder andother neurodevelopmental disorders, Down's Syndrome, Multiple Sclerosis,Amyotrophic lateral sclerosis, brain cancer, encephalitis, infection,trauma, stroke, and paralysis.

In an embodiment, the BMPS includes two or more neuronal cell types thatinclude one or more genetically modified cells. The BMPS wherein the oneor more genetically modified cells include one or more reporter genes.The BMPS further comprises one or more endothelial cells capable offorming a blood-brain-barrier.

In an embodiment, the synthetic neurological organ may include two ormore neural cell types that include one or more genetically modifiedcells. The synthetic neurological organ including one or moregenetically modified cells that include one or more reporter genes. Thesynthetic neurological organ further comprising one or more endothelialcells capable of forming a blood-brain-barrier.

In an aspect, the disclosure provides a method of reproducibly producingan in vitro brain microphysiological system (BMPS), comprising: exposingone or more NPC types to gyratory shaking or stirring; anddifferentiating the one or more NPC types into one or more neural celltypes aggregated into a spheroid mass, wherein the spheroid mass has adiameter that is less than 500 μm.

In an embodiment, the spheroid mass has a diameter that is less thanabout 450 μm, 400 μm, 350 μm, or 300 μm, or a diameter that is betweenabout 350 μm and about 300 μm, or a diameter that is between about 330μm and about 300 μm, or a diameter that is about 310 μm.

In an embodiment, the two or more neural cell types of the in vitro BMPSexpress one or more biomarker selected from the group consisting ofGRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII,NEUN, SLC1A6, and any combination thereof.

In an embodiment, the two or more neural cell types of the in vitro BMPSexpress one or more biomarker selected from the group consisting ofGRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII,NEUN, SLC1A6, and any combination thereof.

In an embodiment, the two or more neural cell types of the in vitro BMPSexpress one or more biomarker selected from the group consisting ofGRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2, FOXO4, CNP, MBP, TH, TUBIII,NEUN, SLC1A6, and any combination thereof.

In an embodiment, inducing comprises a single PSC.

In an embodiment, the an in vitro brain microphysiological system (BMPS)may be produced according to the above described method.

It is also contemplated within the scope of the invention that theaddition of other cells inside (see e.g., FIG. 6) and outside (see e.g.,FIG. 7) the BMPS may be used to modify the structure/composition of theBMPS, such as, e.g., by forming a blood-brain-barrier. It is alsocontemplated that the BMPS described herein may include geneticallymodified pluripotent stem cells, or be combined with other organoids(see e.g., Example 11).

Definitions

By “agent” is meant any small compound, antibody, nucleic acid molecule,or polypeptide, or fragments thereof.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10% change in expression levels,preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

In this disclosure, “comprises,” “comprising,” “containing,” and“having” and the like may have the meaning ascribed to them in U.S.Patent law and may mean “includes,” “including,” and the like;“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “effective amount” is meant the amount of an agent needed toameliorate the symptoms of a neurological disease relative to anuntreated patient. The effective amount of active agent(s) used topractice the present invention for therapeutic treatment of aneurological disease varies depending upon the manner of administration,the age, body weight, and general health of the subject. Ultimately, theattending physician or veterinarian will decide the appropriate amountand dosage regimen. Such amount is referred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000 nucleotides or amino acids, or more.

By “gene” is meant a locus (or region) of DNA that encodes a functionalRNA or protein product, and is the molecular unit of heredity.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “modulate” is meant alter (increase or decrease). Such alterationsare detected by standard art known methods such as those describedherein.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aswell as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

By “pluripotency” is meant stem cells with the potential todifferentiate into any of the three germ layers: endoderm (e.g.,interior stomach lining, gastrointestinal tract, the lungs), mesoderm(e.g., muscle, bone, blood, urogenital), or ectoderm (e.g., epidermaltissues and nervous system). However, one of skill in the art willunderstand that cell pluripotency is a continuum, ranging from thecompletely pluripotent cell that can form every cell of the embryoproper, e.g., embryonic stem cells and iPSCs (see below), to theincompletely or partially pluripotent cell that can form cells of allthree germ layers but that may not exhibit all the characteristics ofcompletely pluripotent cells. Induced pluripotent stem cells, commonlyabbreviated as iPS cells or iPSCs are a type of pluripotent stem cellartificially derived from a non-pluripotent cell, typically an adultsomatic cell, by inducing a “forced” expression of certain genes andtranscription factors. These transcription factors play a key role indetermining the state of these cells and also highlight the fact thatthese somatic cells do preserve the same genetic information as earlyembryonic cells. The ability to induce cells into a pluripotent statewas initially pioneered using mouse fibroblasts and four transcriptionfactors, Oct4, Sox2, Klf4 and c-Myc;—a process called reprogramming. Thesuccessful induction of human iPSCs derived from human dermalfibroblasts has been performed using methods similar to those used forthe induction of mouse cells. These induced cells exhibit similar traitsto those of embryonic stem cells (ESCs) but do not require the use ofembryos. Some of the similarities between ESCs and iPSCs includepluripotency, morphology, self-renewal ability, a trait that impliesthat they can divide and replicate indefinitely, and gene expression.

By “stem cells” is meant undifferentiated biological cells that candifferentiate into specialized cells and can divide (through mitosis) toproduce more stem cells. They are found in multicellular organisms. Inmammals, there are two broad types of stem cells: embryonic stem cells,which are isolated from the inner cell mass of blastocysts, and adultstem cells, which are found in various tissues. In adult organisms, stemcells and progenitor cells act as a repair system for the body,replenishing adult tissues. In a developing embryo, stem cells candifferentiate into all the specialized cells—ectoderm, endoderm andmesoderm (see induced pluripotent stem cells)—but also maintain thenormal turnover of regenerative organs, such as blood, skin, orintestinal tissues. There are three known accessible sources ofautologous adult stem cells in humans: 1. Bone marrow, which requiresextraction by harvesting, that is, drilling into bone (typically thefemur or iliac crest). 2. Adipose tissue (lipid cells), which requiresextraction by liposuction. 3. Blood, which requires extraction throughapheresis, wherein blood is drawn from the donor (similar to a blooddonation), and passed through a machine that extracts the stem cells andreturns other portions of the blood to the donor. Stem cells can also betaken from umbilical cord blood just after birth. Of all stem celltypes, autologous harvesting involves the least risk. By definition,autologous cells are obtained from one's own body.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a neurological disorder and/orsymptoms associated therewith. It will be appreciated that, although notprecluded, treating a disorder or condition does not require that thedisorder, condition or symptoms associated therewith be completelyeliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “an,” and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

A “therapeutically effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. An effectiveamount can be administered in one or more administrations.

By “GRIN1 polypeptide” (or glutamate ionotropic receptor NMDA typesubunit 1) is meant a polypeptide or fragment thereof having at leastabout 85% amino acid identity to NCBI Accession No. Q05586.

  1 mstmrlltla llfscsvara acdpkivnig avlstrkheq mfreavnqan krhgswkiql 61 natsvthkpn aiqmalsvce dlissqvyai lvshpptpnd hftptpvsyt agfyripvlg121 lttrmsiysd ksihlsflrt vppyshqssv wfemmrvysw nhiillvsdd hegraaqkrl181 etlleeresk aekvlqfdpg tknvtallme akelearvii lsaseddaat vyraaamlnm241 tgsgyvwlvg ereisgnalr yapdgilglq lingknesah isdavgvvaq avhelleken301 itdpprgcvg ntniwktgpl fkrvlmssky adgvtgrvef nedgdrkfan ysimnlqnrk361 lvqvgiyngt hvipndrkii wpggetekpr gyqmstrlki vtihqepfvy vkptlsdgtc421 keeftvngdp vkkvictgpn dtspgsprht vpqccygfci dlliklartm nftyevhlva481 dgkfgtqerv nnsnkkewng mmgellsgqa dmivapltin neraqyiefs kpfkyqglti541 lvkkeiprst ldsfmqpfqs tlwllvglsv hvvavmlyll drfspfgrfk vnseeeeeda601 ltlssamwfs wgvllnsgig egaprsfsar ilgmvwagfa miivasytan laaflvldrp661 eeritgindp rlrnpsdkfi yatvkqssvd iyfrrqvels tmyrhmekhn yesaaeaiqa721 vrdnklhafi wdsavlefea sqkcdlvttg elffrsgfgi gmrkdspwkq nvslsilksh781 engfmedldk twvryqecds rsnapatltf enmagvfmlv aggivagifl ifieiaykrh841 kdarrkqmql afaavnvwrk nlqdrksgra epdpkkkatf raitstlass fkrrrsskdt901 stgggrgalq nqkdtvlprr aiereegqlq lcsrhres

By “GRIN1 nucleic acid molecule” (or glutamate ionotropic receptor NMDAtype subunit 1) is meant a polynucleotide encoding an GRIN1 polypeptide.An exemplary GRIN1 nucleic acid molecule (e.g., mRNA) is provided atNCBI Accession No. NM_007327.

   1 gtcgccgcag cgtccggacc ggaaccagcg ccgtccgcgg agccgccgcc gccgccgccg  61 ggccctttcc aagccgggcg ctcggagctg tgcccggccc cgcttcagca ccgcggacag 121 cgccggccgc gtggggctga gccccgagcc cccgcgcacg cttcagcgcc ccttccctcg 181 gccgacgtcc cgggaccgcc gctccggggg agacgtggcg tccgcagccc gcggggccgg 241 gcgagcgcag gacggcccgg aagccccgcg ggggatgcgc cgagggcccc gcgttcgcgc 301 cgcgcagagc caggcccgcg gcccgagccc atgagcacca tgcgcctgct gacgctcgcc 361 ctgctgttct cctgctccgt cgcccgtgcc gcgtgcgacc ccaagatcgt caacattggc 421 gcggtgctga gcacgcggaa gcacgagcag atgttccgcg aggccgtgaa ccaggccaac 481 aagcggcacg gctcctggaa gattcagctc aatgccacct ccgtcacgca caagcccaac 541 gccatccaga tggctctgtc ggtgtgcgag gacctcatct ccagccaggt ctacgccatc 601 ctagttagcc atccacctac ccccaacgac cacttcactc ccacccctgt ctcctacaca 661 gccggcttct accgcatacc cgtgctgggg ctgaccaccc gcatgtccat ctactcggac 721 aagagcatcc acctgagctt cctgcgcacc gtgccgccct actcccacca gtccagcgtg 781 tggtttgaga tgatgcgtgt ctacagctgg aaccacatca tcctgctggt cagcgacgac 841 cacgagggcc gggcggctca gaaacgcctg gagacgctgc tggaggagcg tgagtccaag 901 gcagagaagg tgctgcagtt tgacccaggg accaagaacg tgacggccct gctgatggag 961 gcgaaagagc tggaggcccg ggtcatcatc ctttctgcca gcgaggacga tgctgccact1021 gtataccgcg cagccgcgat gctgaacatg acgggctccg ggtacgtgtg gctggtcggc1081 gagcgcgaga tctcggggaa cgccctgcgc tacgccccag acggcatcct cgggctgcag1141 ctcatcaacg gcaagaacga gtcggcccac atcagcgacg ccgtgggcgt ggtggcccag1201 gccgtgcacg agctcctcga gaaggagaac atcaccgacc cgccgcgggg ctgcgtgggc1261 aacaccaaca tctggaagac cgggccgctc ttcaagagag tgctgatgtc ttccaagtat1321 gcggatgggg tgactggtcg cgtggagttc aatgaggatg gggaccggaa gttcgccaac1381 tacagcatca tgaacctgca gaaccgcaag ctggtgcaag tgggcatcta caatggcacc1441 cacgtcatcc ctaatgacag gaagatcatc tggccaggcg gagagacaga gaagcctcga1501 gggtaccaga tgtccaccag actgaagatt gtgacgatcc accaggagcc cttcgtgtac1561 gtcaagccca cgctgagtga tgggacatgc aaggaggagt tcacagtcaa cggcgaccca1621 gtcaagaagg tgatctgcac cgggcccaac gacacgtcgc cgggcagccc ccgccacacg1681 gtgcctcagt gttgctacgg cttttgcatc gacctgctca tcaagctggc acggaccatg1741 aacttcacct acgaggtgca cctggtggca gatggcaagt tcggcacaca ggagcgggtg1801 aacaacagca acaagaagga gtggaatggg atgatgggcg agctgctcag cgggcaggca1861 gacatgatcg tggcgccgct aaccataaac aacgagcgcg cgcagtacat cgagttttcc1921 aagcccttca agtaccaggg cctgactatt ctggtcaaga aggagattcc ccggagcacg1981 ctggactcgt tcatgcagcc gttccagagc acactgtggc tgctggtggg gctgtcggtg2041 cacgtggtgg ccgtgatgct gtacctgctg gaccgcttca gccccttcgg ccggttcaag2101 gtgaacagcg aggaggagga ggaggacgca ctgaccctgt cctcggccat gtggttctcc2161 tggggcgtcc tgctcaactc cggcatcggg gaaggcgccc ccagaagctt ctcagcgcgc2221 atcctgggca tggtgtgggc cggctttgcc atgatcatcg tggcctccta caccgccaac2281 ctggcggcct tcctggtgct ggaccggccg gaggagcgca tcacgggcat caacgaccct2341 cggctgagga acccctcgga caagtttatc tacgccacgg tgaagcagag ctccgtggat2401 atctacttcc ggcgccaggt ggagctgagc accatgtacc ggcatatgga gaagcacaac2461 tacgagagtg cggcggaggc catccaggcc gtgagagaca acaagctgca tgccttcatc2521 tgggactcgg cggtgctgga gttcgaggcc tcgcagaagt gcgacctggt gacgactgga2581 gagctgtttt tccgctcggg cttcggcata ggcatgcgca aagacagccc ctggaagcag2641 aacgtctccc tgtccatcct caagtcccac gagaatggct tcatggaaga cctggacaag2701 acgtgggttc ggtatcagga atgtgactcg cgcagcaacg cccctgcgac ccttactttt2761 gagaacatgg ccggggtctt catgctggta gctgggggca tcgtggccgg gatcttcctg2821 attttcatcg agattgccta caagcggcac aaggatgctc gccggaagca gatgcagctg2881 gcctttgccg ccgttaacgt gtggcggaag aacctgcagg atagaaagag tggtagagca2941 gagcctgacc ctaaaaagaa agccacattt agggctatca cctccaccct ggcttccagc3001 ttcaagaggc gtaggtcctc caaagacacg agcaccgggg gtggacgcgg cgctttgcaa3061 aaccaaaaag acacagtgct gccgcgacgc gctattgaga gggaggaggg ccagctgcag3121 ctgtgttccc gtcataggga gagctgagac tccccgcccg ccctcctctg ccccctcccc3181 cgcagacaga cagacagacg gacgggacag cggcccggcc cacgcagagc cccggagcac3241 cacggggtcg ggggaggagc acccccagcc tcccccaggc tgcgcctgcc cgcccgccgg3301 ttggccggct ggccggtcca ccccgtcccg gccccgcgcg tgcccccagc gtggggctaa3361 cgggcgcctt gtctgtgtat ttctattttg cagcagtacc atcccactga tatcacgggc3421 ccgctcaacc tctcagatcc ctcggtcagc accgtggtgt gaggcccccg gaggcgccca3481 cctgcccagt tagcccggcc aaggacactg atgggtcctg ctgctcggga aggcctgagg3541 gaagcccacc cgccccagag actgcccacc ctgggcctcc cgtccgtccg cccgcccacc3601 ccgctgcctg gcgggcagcc cctgctggac caaggtgcgg accggagcgg ctgaggacgg3661 ggcagagctg agtcggctgg gcagggccgc agggcgctcc ggcagaggca gggccctggg3721 gtctctgagc agtggggagc gggggctaac tggccccagg cggaggggct tggagcagag3781 acggcagccc catccttccc gcagcaccag cctgagccac agtggggccc atggccccag3841 ctggctgggt cgcccctcct cgggcgcctg cgctcctctg cagcctgagc tccaccctcc3901 cctcttcttg cggcaccgcc cacccacacc ccgtctgccc cttgacccca cacgccgggg3961 ctggccctgc cctcccccac ggccgtccct gacttcccag ctggcagcgc ctcccgccgc4021 ctcgggccgc ctcctccaga ctcgagaggg ctgagcccct cctctcctcg tccggcctgc4081 agcccagaac gggcctcccc gggggtcccc ggacgctggc tcgggactgt cttcaaccct4141 gccctgcacc ttgggcacgg gagagcgcca cccgcccgcc cccgccctcg ctccgggtgc4201 gtgaccggcc cgccaccttg tacagaacca gcactcccag ggcccgagcg cgtgccttcc4261 ccgtgcggcc cgtgcgcagc cgcgctctgc ccctccgtcc ccagggtgca ggcgcgcacc4321 gcccaacccc cacctcccgg tgtatgcagt ggtgatgcct aaaggaatgt cacgcagttt4381 tcaaaaaaaa aaaaaaaaaa

By “GAD1 polypeptide” (or glutamate decarboxylase 1) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. Q99259.

  1 masstpsssa tssnagadpn ttnlrpttyd twcgvahgct rklglkicgf lqrtnsleek 61 srlvsafker qssknllsce nsdrdarfrr tetdfsnlfa rdllpaknge eqtvqfllev121 vdillnyvrk tfdrstkvld fhhphqlleg megfnlelsd hpesleqilv dcrdtlkygv181 rtghprffnq lstgldiigl agewltstan tnmftyeiap vfvlmeqitl kkmreivgws241 skdgdgifsp ggaisnmysi maarykyfpe vktkgmaavp klvlftseqs hysikkagaa301 lgfgtdnvil ikcnergkii padfeakile akqkgyvpfy vnatagttvy gafdpiqeia361 dicekynlwl hvdaawgggl lmsrkhrhkl ngieransvt wnphkmmgvl lqcsailvke421 kgilqgcnqm cagylfqpdk qydvsydtgd kaiqcgrhvd ifkfwlmwka kgtvgfenqi481 nkclelaeyl yakiknreef emvfngepeh tnvcfwyipq slrgvpdspq rreklhkvap541 kikalmmesg ttmvgyqpqg dkanffrmvi snpaatqsdi dflieeierl gqdl

By “GAD1 nucleic acid molecule” (or glutamate decarboxylase 1) is meanta polynucleotide encoding an GAD1 polypeptide. An exemplary GAD1 nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. BC036552.

   1 agcgtgtggt agaggagaaa cgctgaaacc ggaccgaaac ctcgccctag gcttagcgat  61 ggctaaaaac cggctgggac aagagggagg caagcaacat tccgactcgc tgctttctgg 121 ctgtctggag tgcaaggtga ctgtggttct tctctggcca agtccgaggg agaacgtaaa 181 gatatgggcc tttttccccc tctcaccttg tctcaccaaa gtccctagtc cccggagcag 241 ttagcctctt tctttccagg gaattagcca gacacaacaa cgggaaccag acaccgaacc 301 agacatgccc gccccgtgcg ccctcccccc gctggcccac acgccggctg ctgagtgccc 361 aatggggctt gtagcggctc ggctggaaaa tcgctcactg agcgctcccc tgtgctccta 421 gcccagtccc ccacaccctt gcgtcttgta ctggccttgg acccccaccc cgaccccgac 481 cccgcctcgt ctcggcgctt cactccaggt cgcgccgatg caccgccaga ctcgagagcg 541 gcccagggct acgctccctg cgccccagta ccggagctag cgcgcacgtc tcctccgctg 601 cccccacccc tgcgcacccc taccaggcag gctcgctgcc tttcctccct cttgtctctc 661 cagagccgga tcttcaaggg gagcctccgt gcccccggct gctcagtccc tccggtgtgc 721 aggaccccgg aagtcctccc cgcacagctc tcgcttctct ttgcagcctg tttctgcgcc 781 ggaccagtcg aggactctgg acagtagagg ccccgggacg accgagctga tggcgtcttc 841 gaccccatct tcgtccgcaa cctcctcgaa cgcgggagcg gaccccaata ccactaacct 901 gcgccccaca acgtacgata cctggtgcgg cgtggcccat ggatgcacca gaaaactggg 961 gctcaagatc tgcggcttct tgcaaaggac caacagcctg gaagagaaga gtcgccttgt1021 gagtgccttc aaggagaggc aatcctccaa gaacctgctt tcctgtgaaa acagcgaccg1081 ggatgcccgc ttccggcgca cagagactga cttctctaat ctgtttgcta gagatctgct1141 tccggctaag aacggtgagg agcaaaccgt gcaattcctc ctggaagtgg tggacatact1201 cctcaactat gtccgcaaga catttgatcg ctccaccaag gtgctggact ttcatcaccc1261 acaccagttg ctggaaggca tggagggctt caacttggag ctctctgacc accccgagtc1321 cctggagcag atcctggttg actgcagaga caccttgaag tatggggttc gcacaggtca1381 tcctcgattt ttcaaccagc tctccactgg attggatatt attggcctag ctggagaatg1441 gctgacatca acggccaata ccaacatgtt tacatatgaa attgcaccag tgtttgtcct1501 catggaacaa ataacactta agaagatgag agagatagtt ggatggtcaa gtaaagatgg1561 tgatgggata ttttctcctg ggggcgccat atccaacatg tacagcatca tggctgctcg1621 ctacaagtac ttcccggaag ttaagacaaa gggcatggcg gctgtgccta aactggtcct1681 cttcacctca gaacagagtc actattccat aaagaaagct ggggctgcac ttggctttgg1741 aactgacaat gtgattttga taaagtgcaa tgaaaggggg aaaataattc cagctgattt1801 tgaggcaaaa attcttgaag ccaaacagaa gggatatgtt cccttttatg tcaatgcaac1861 tgctggcacg actgtttatg gagcttttga tccgatacaa gagattgcag atatatgtga1921 gaaatataac ctttggttgc atgtcgatgg atttaacttc tcacaattgg ccaataggat1981 catctgcctt gctactgaac taatgactaa caaaggctgt gtcacgtggc atcccaacta2041 ttcagtaaac atgcatcatg gctgcctggg gaggtgggct gctcatgtcc aggaagcacc2101 accataaact caacggcata gaaagggcca actcagtcac ctggaaccct cacaagatga2161 tgggcgtgct gttgcagtgc tctgccattc tcgtcaagga aaagggtata ctccaaggat2221 gcaaccagat gtgtgcagga tacctcttcc agccagacaa gcagtatgat gtctcctacg2281 acaccgggga caaggcaatt cagtgtggcc gccacgtgga tatcttcaag ttctggctga2341 tgtggaaagc aaagggcaca gtgggatttg aaaaccagat caacaaatgc ctggaactgg2401 ctgaatacct ctatgccaag attaaaaaca gagaagaatt tgagatggtt ttcaatggcg2461 agcctgagca cacaaacgtc tgtttttggt atattccaca aagcctcagg ggtgtgccag2521 acagccctca acgacgggaa aagctacaca aggtggctcc aaaaatcaaa gccctgatga2581 tggagtcagg tacgaccatg gttggctacc agccccaagg ggacaaggcc aacttcttcc2641 ggatggtcat ctccaaccca gccgctaccc agtctgacat tgacttcctc attgaggaga2701 tagaaagact gggccaggat ctgtaatcat ccttcgcaga acatgagttt atgggaatgc2761 cttttccctc tggcactcca gaacaaacct ctatatgttg ctgaaacaca caggccattt2821 cattgaggga aaacataata tcttgaagaa tattgttaaa accttactta aagcttgttt2881 gttctagtta gcaggaaata gtgttctttt taaaaagttg cacattagga acagagtata2941 tatgtacagt tatacatacc tctctctata tatacatgta tagtgagtgt ggcttagtaa3001 tagatcacgg catgtttccc gctccaagag aattcacttt accttcagca gttaccgagg3061 agctaaacat gctgccaacc agcttgtcca acaactccag gaaaactgtt tttcaaaacg3121 ccatgtccta ggggccaagg gaaatgctgt tggtgagaat cgacctcact gtcagcgttt3181 ctccacctga agtgatgatg gatgagaaaa aacaccacca aatgacaagt cacaccctcc3241 ccattagtat cctgttaggg gaaaatagta gcagagtcat tgttacaggt gtactatggc3301 tgtattttta gagattaatt tgtgtagatt gtgtaaattc ctgttgtctg accttggtgg3361 tgggaggggg agactatgtg tcatgatttc aatgattgtt taattgtagg tcaatgaaat3421 atttgcttat ttatattcag agatgtacca tgttaaagag gcgtcttgta ttttcttccc3481 atttgtaatg tatcttattt atatatgaag taagttctga aaactgttta tggtattttc3541 gtgcatttgt gagccaaaga gaaaagatta aaattagtga gatttgtatt tatattagag3601 tgcccttaaa ataatgattt aagcatttta ctgtctgtaa gagaattcta agattgtaca3661 taaagtcata tatatggaaa tcctgttact taaatagcat ctgctcttct cttacgctct3721 ctgtctggct gtacgtctgg tgttctcaat gcttttctag caactgttgg ataataacta3781 gatctcctgt aattttgtag tagttgatga ccaatctctg ttactcgctt agctgaaacc3841 taaggcaaca tttccgaaga ccttctgaag atctcagata aagtgaccag gctcacaact3901 gtttttgaag aagggaaatt cacactgtgc gttttagagt atgcaagaag aatataaata3961 aataaaaata ttctccatgg agaatttgaa caaaaaaaaa aaaaaaa

By “GABA polypeptide” (or gamma-Aminobutyric acid) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. P30531.

  1 matngskvad gqistevsea pvandkpktl vvkvqkkaad lpdrdtwkgr fdflmscvgy 61 aiglgnvwrf pylcgknggg aflipyfltl ifagvplfll ecslgqytsi gglgvwklap121 mfkgvglaaa vlsfwlniyy iviiswaiyy lynsftttlp wkqcdnpwnt drcfsnysmv181 nttnmtsavv efwernmhqm tdgldkpgqi rwplaitlai awilvyfciw kgvgwtgkvv241 yfsatypyim liilffrgvt lpgakegilf yitpnfrkls dsevwldaat qiffsyglgl301 gslialgsyn sfhnnvyrds iivccinsct smfagfvifs ivgfmahvtk rsiadvaasg361 pglaflaype avtqlpispl wailffsmll mlgidsqfct vegfitalvd eyprllrnrr421 elfiaavcii syliglsnit qggiyvfklf dyysasgmsl lflvffecvs iswfygvnrf481 ydniqemvgs rpciwwklcw sfftpiivag vfifsavqmt pltmgnyvfp kwgqgvgwlm541 alssmvlipg ymaymfltlk gslkqriqvm vqpsedivrp engpeqpqag sstskeayi

By “GABA nucleic acid molecule” (or gamma-Aminobutyric acid) is meant apolynucleotide encoding an GABA polypeptide. An exemplary GABA nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. U76343.

   1 gtagcttcac taaggtggga tggatagcag ggtctcaggc acaaccagta atggagagac  61 aaaaccantg tatcacaaga tggagtttgt gctgtcagtg gctggggaga tcattggctt 121 aggcaacgtc tggaggtttc cctatctctg ctacaaaaat gggggaggtg ccttcttcat 181 cccctacctc gtcttcctct ttacctgtgg cattcctgtc ttccttctgg agacagcact 241 aggccagtac actagccagg gaggcgtcac agcctggagg aagatctgcc ccatctttga 301 gggcattggc tatgcctccc agatgatcgt catcctcctc aacgtctact acatcattgt 361 gttggcctgg gccctgttct acctcttcag cagcttcacc atcgacctgc cctggggcgg 421 ctgctaccat gagtggaaca cagaacactg tatggagttc cagaagacca acggctccct 481 gaatggtacc tctgagaatg ccacctctcc tgtcatcgag ttctgggagc ggcgggtctt 541 gaagatctct gatgggatcc agcacctggg ggccctgcgc tgggagctgg ctctgtgcct 601 cctgctggcc tgggtcatct gctacttctg catctggaag ggggtgaagt ccacaggcaa 661 ggtggtgtac ttcacggcca catttcctta cctcatgctg gtggtcctgt taattcgagg 721 ggtgacgttg cctggggcag cccaaggaat tcagttttac ctgtacccaa acctcacgcg 781 tctgtgggat ccccaggtgt ggatggatgc aggcacccag atattcttct ccttcgccat 841 ctgtcttggg tgcctgacag ccctgggcag ctacaacaag taccacaaca actgctacag 901 cggcaccagc tttgtggccg gctttgccat cttctccatc ctgggcttca tgtctcagga 961 gcagggggtg cccatttctg aggtggccga gtcaggccct ggcctggctt tcatcgctta1021 cccgcgggct gtggtgatgc tgcccttctc tcctctctgg gcctgctgtt tcttcttcat1081 ggtcgttctc ctgggactgg atagccagtt tgtgtgtgta gaaagcctgg tgacagcgct1141 ggtggacatg taccctcacg tgttccgcaa gaagaaccgg agggaagtcc tcatccttgg1201 agtatctgtc gtctccttcc ctgtggggct gatcatgctc acagagggcg gaatgtacgt1261 gttccagctc tttgactact atgcggccag tggcatgtgc ctcctgttcg tggccatctt1321 cgagtccctc tgtgtggctt gggtttacgg agccaagcgc ttctacgaca acatcgaaga1381 catgattggg tacaggccat ggcctcttat caaatactgt tggctcttcc tcacaccagc1441 tgtgtgcaca gccacctttc tcttctccct gataaagtac actccgctga cctacaacaa1501 gaagtacacg tacccgtggt ggggcgatgc cctgggctgg ctcctggctc tgtcctcctg1561 gtctgcattc ctgcctggag cctctacaga ctcggaaccc tcaagggccc cttcagagag1621 agaatccgtc agctcatgtg cccagccgag gacctgcccc agcggaaccc agcaggaccc1681 tcggctcccg ccacccccag gacctcactg ctcagactca cagagctaga gtctcactgc1741 tagggggcag gcccttggat ggtgcctgtg tgcctggcct tggggatggc tgtggaggga1801 acgtggcaga agcagcccca tgtgttccct gcccccgacc tggagtggat aagacaagag1861 gggtattttg gagtccacct gctgagctgg aggcctccca ctgcaacttt tcagctcagg1921 ggttgttgaa cagatgtgaa aaggccagtg ccaagagtgt ccctcggaga cccttgaagg1981 c

By “TH polypeptide” (or Tyrosine Hydroxylase) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. NP_002692.

  1 mptpdattpq akgfrravse ldakqaeaim vrgqgapgps ltgspwpgta apaasytptp 61 rsprfigrrq sliedarker eaavaaaaaa vpsepgdple avafeekegk avlnllfspr121 atkpsalsra vkvfetfeak ihhletrpaq rpraggphle yfvrlevrrg dlaallsgvr181 qvsedvrspa gpkvpwfprk vseldkchhl vtkfdpdldl dhpgfsdqvy rqrrkliaei241 afqyrhgdpi prveytaeei atwkevyttl kglyathacg ehleafalle rfsgyredni301 pqledvsrfl kertgfqlrp vagllsardf laslafrvfq ctqyirhass pmhspepdcc361 hellghvpml adrtfaqfsq diglaslgas deeieklstl ywftvefglc kqngevkayg421 agllssygel lhclseepei rafdpeaaav qpyqdqtyqs vyfvsesfsd akdklrsyas481 riqrpfsvkf dpytlaidvl dspqavrrsl egvqdeldtl ahalsaig

By “TH nucleic acid molecule” (or Tyrosine Hydroxylase) is meant apolynucleotide encoding an TH polypeptide. An exemplary TH nucleic acidmolecule is provided at NCBI Accession No. NG_008128.

    1 gcgggggggc agtgtgtgct ccagcatgtg tgtgtgtgtg tgcatgtaca cgtgtgcacc   61 tgtatcgcct gtgtgtgtgc atgtgatgtg tacacgtgtc atgcatgcac gcacatgtgt  121 agtgtgtgct cgtgtgtggt gtgtgcctgt gtcatgtatg agcacacttg tatatgttgt  181 gtgtactgtg tcatatatga gtgtgtttgc ctgtgtagtg catgcacatc cgtgtgtgca  241 tctggtgtgt ccgtgggtca ttacgagtgc atcgtatgtg tatcgtgtac atgagtacac  301 ttgtatgtgt ggtgtgtaca ggtgccatgt aagtgtgctt gtacatatat gcatgcatgt  361 gtcatatgca tctgtgtgtg catgtgtgtg gtgcacacat gtgttatgtc tgagtgtgcc  421 tgtatgtgtg ctatgtacac gtcatgtgtg agtgtgcttg catgtgcagt gtgtggatgc  481 tgcttgtacc tgtggtgtgt acctgtgtca tgggtgctca cacgtgcatg gagtgttgtg  541 tgtgtgcttg tgtgccccat gtgtgcatgt gtgtgtgcct cacacagatg cctgcatttg  601 cctaggcact tgcaagagga caccatgctg gctctcaaag atcacagggc cacctgagcc  661 ctgtgcacac cacagccagg ccatggctag accctgcaga gccacagggc gatgcctgtc  721 agccagggga cccagaacac ctcctgggct cctccccagc acatggctgg gctcctccag  781 caggcctgga tttgggaagg gcccgtggtg ggcaaggctg gtgctgggga gcaggcctgg  841 tggcctcaga gactcgccct gtgggcggag cagcctcaca gccaggtcga agtcagcact  901 ctgaccctgc cccacgcggg gagtgggcac cagtcccagg gcacagacgt gctgggtgat  961 taatctgggt gattaagcct cgggctgaga ggctgttgag agagaacacg ctccattgtg 1021 gagctggctc agcattcctt acggccatgg tggcaggggc tgtaaccaca gggacggcgg 1081 aagtggtgga gggtggtggg gtatggaggg aagcccagag ggctccgtgc aggaaggtgg 1141 agcctggtgc aatggagggg acagcaaggg ctcctcagac ctctgcgggg cccccactcc 1201 cctggtcacc tgttttgtct ctgatctggc ctgggtcggc cctcactcct ggccccacct 1261 catagccccc cctggtgggg ctccgctcca gcccttctcc ttcccagggg ccagtatgct 1321 ggccccaggg gtctcttggg gcgtgacctc ggcctccaga gaaccctgtc ccagctctgc 1381 ccttccctct ggggtctctg tagatgggac gctggtcaca gcagcctgtc tgatttgttc 1441 cctgtggcct aggttcctga gccccacagt gccaggggat ggatgccacc ggatctttga 1501 aagaccagtg tcaggccggg cgcagtggct cacgcctgta atcccagcac tttgggaggc 1561 cgaggtgggc ggatcacgaa gtcaggagat cgagaccatc ctggctaaca cagtgaaacc 1621 ccgtctccac taaaaataca aaaagttagc tgggcgtggt ggtgggcgcc tgtagtccca 1681 gctacttggg aggctgaggc aggagaatgg cgtgaaccgg ggaggcggag cttgcagtga 1741 gccgagatcg cgccattgca ctccagcctg ggtgacagag cgagactcgg tctcaaaaaa 1801 aaagaaaaaa aggaaagacc agtgtcttgg gagttgggaa acctgggctg gagactcact 1861 gcatgacccc tgagaagttg cacctcagaa cctcagtcct cgcatctgca gaatgggtct 1921 gtgaacacct cagctgcccg aacgtggatg ccgcaggctg acccagcact gagctctacc 1981 aagaccaggg gccagccgtg tgctccctcc aggcctgtgc ccagcgtgga gaggcctcgt 2041 cccgtgggcg ctggagtgga gccttcctgg tgtttgtgga catctctgga gagggccaga 2101 ggcaggtggg tgacacgggg catggctcaa tcatgggtgg tccagactgg agaggtaccc 2161 tcgggctggg agcggggagg ctggccaggg tagacttttg gggcctccat ggataccctc 2221 accatctgga atcggagagg ggcacggcac aaaggagggc ggggccaggg ccaggactgg 2281 agtcgggggc acctctgtgc caacaggggc cttggatctg gggtacagca tggttccccg 2341 gccctgaagg ggctggcgtg tgggacaggc ttcccaggaa tggataggca gggatggatg 2401 ctgcctgatt ggggcgggag gctggaggca gggcaggtgc aggcacctga gggcagcact 2461 cacctccaca ggggtccagg ggcctcccca gcctcagcac ctggcctggg ctcctgcctc 2521 cagagagcct ggccccaagg aagagtctag taagcttagt tcccatcggg cttccatgaa 2581 agcacaactg gcccggcagg aaaccgaatt aaaaagcaat atttgtatca gtggaagaca 2641 tttgctgaaa ggttaaatcc acatccggca gtgtgggcca tgagcctccg gcgtggtgtt 2701 catcaggcat gtctctcctc ctggcctggg cacctgagca ctggggccgc cctgggcaga 2761 gctggggcgg ggtgctgggg ggcctggagc tgcctcaccg agggatcctc agcagccgac 2821 cctgggggag gcaaatgaga ctctttctgg ggaccttgag gggagctcgg gggagccatg 2881 cagagcttca ccaggcctgg acactgggca tggaggctgg gccacccaag ggccatcacc 2941 agggactcag gtgggtgggc ctcagccctg ggtgacagaa gctcacgggc cgcagggcga 3001 ggccagaggc tgagccttca ggctgaggtc ttggaggcaa atccctccaa cgcccttctg 3061 agcaggcacc cagacctact gtgggcagga cccacaggag gtggaggcct ttggggaaca 3121 ctgtggaggg gcatagcatc tccgagagag gacagggtct gcactgggtg ctgagagaca 3181 gcaggggccg agcggtaggc ttccctgccc ccagggatgt tccagaggag cgcaagggag 3241 gggcattaat atcgtggcaa gaaagggcag gcattgcaga gtgagcagcg acggaactgg 3301 gttttgtggg atgcatagga gttcacccgg ataagaggtg ggtgaggaat gacactgcaa 3361 accggggatc acggagcccc aaatccttct gggccaggaa gtgggaaggg ttggggggtc 3421 ttccctttgc tttgactgag cactcagcct gcctgcagag ggcagcgagg agccacggag 3481 gggtgtggga cagggatgcc atggctgaag cagttttagg aaaggtccca ggggctattg 3541 ttgaagagag aacggggagc ggggagtccc acagctgaca ggagcagagt gggccctgag 3601 agatgccagc tctggctgcc acagtgacca gccggggtag gccttcgaga agtcagggag 3661 cgtctagggc ttctggctcc tgctgggccc agggtgtcat cttgggctgc caacaccaga 3721 aagcccagca gatacaggaa gccccaagcc ctgtcggaaa cggttcttct ccaggaggga 3781 cagcggtggc agcgttcagc cgcaggccat gcactctggg gccacgtcct tccctctgta 3841 cagtccagca ttgtcaaggc aggctctggc catctctgct gaccccagag ggatggggag 3901 gcctcccctt ccaccagaag ggccagaagc caccctgggc aggggcatca ctctccctgg 3961 gtggggcagc ggctgggagc aggaggtgcc agtgggcgtg ggctggatgc gggtgcctgc 4021 ggggcggaca tggaacttgg gggaggctct aggctggggt tgtcctcaag ggagttctca 4081 ggtcacccca gggtcaccct caacccgggg cctggtgggg tagaggagaa actgcaaagg 4141 tctctccaag gggaaggcat cagggccctc agcactgagg gacgtgcgtg ctctttaaag 4201 aaggggccac aggaccccga gggaagccag gagctagcag tgggccatag aggggctgag 4261 tggggtgggt ggaagccgtc cctggccctg gtcgccctgg caaccctggt ggggactgtg 4321 atgcaggagg tggcagccat ttggaaacgc gtggcgtctc cttagagatg tcttcttcag 4381 cctcccaggg tcctccacac tggacaggtg ggccctcctg ggacattctg gaccccacgg 4441 ggcgagcttg ggaagccgct gcaagggcca cacctgcagg gcccgggggc tgtgggcaga 4501 tggcactcct aggaaccacg tctatgagac acacggcctg gaatcttctg gagaagcaaa 4561 caaattgcct cctgacatct gaggctggag gctggattcc ccgtcttggg gctttctggg 4621 tcggtctgcc acgaggttct ggtgttcatt aaaagtgtgc ccctgggctg ccagaaagcc 4681 cctccctgtg tgctctcttg agggctgtgg ggccaagggg accctggctg tctcagcccc 4741 ccgcagagca cgagcccctg gtccccgcaa gcccgcgggc tgaggatgat tcagacaggg 4801 ctggggagtg aaggcaatta gattccacgg acgagccctt tctcctgcgc ctccctcctt 4861 cctcacccac ccccgcctcc atcaggcaca gcaggcaggg gtgggggatg taaggagggg 4921 aaggtggggg acccagaggg ggctttgacg tcagctcagc ttataagagg ctgctgggcc 4981 agggctgtgg agacggagcc cggacctcca cactgagcca tgcccacccc cgacgccacc 5041 acgccacagg ccaagggctt ccgcagggcc gtgtctgagc tggacgccaa gcaggcagag 5101 gccatcatgg taagagggca ggtaggtgcc cggcggccgc agtggaccgg agcccagggc 5161 tggtgccagc tgcctctgct actccccagc ctggctggca gccccaggct cagggtccat 5221 gcaaacccct gggacgcggc gtggatgtgg aggcctgggc acagcggcat cccctgtgcc 5281 tggtgtttga gtccctgttg ggggagggtg aggtgatgcc tgtccctgtg tgtgcccctc 5341 ttaggccgac ctctctcggg ggtcgtgtgg gtctctgtgt cttgtttcat cttgaatctt 5401 aacgatcgga atgtggaaac aaatccatcc aaaaaatcca agatggccag aggtccccgg 5461 ctgctgcacc cagcccccac cctactccca cctgcccctg cctccctctg ccccagctgc 5521 cctagtcagc accccaacca gcctgcctgc ttggggaggc agccccaagg cccttcccag 5581 gctctagcag cagctcatgg tggggggtcc tgggcaaata gggggcaaaa ttcaaagggt 5641 atctgggctc tggggtgatt cccattggcc tgttcctccc ttatttccct cattcattca 5701 ttcattcatt cattcattca ccatggagtc tgtgttccct gtgacctgca ctcggaagcc 5761 ctgtgtacag gggactgtgt gggccaggct ggataatcgg gagcttttca gcccacagga 5821 ggggtcttcg gtgcctcctt gggcactcag aaccttgggc tccctggcac atttaaaatg 5881 ggtttttatt tatggacctt gattgaaatg tggtgtgagt tgtagcagtg tcatttccag 5941 gtaccttctc agggacacag ggcgccctcc cccgtcctcc cccgccctcc cctaccctcc 6001 cccaccaggc tccccatcag gcatcccctc cccagggcgc cccggggccc agcctcacag 6061 gctctccgtg gcctggaact gcagccccag ctgcatccta cacccccacc ccaagggtaa 6121 gtaagagggg actctgggag gggcttctgc tgctcccctt catgttccac aaccctggaa 6181 gctcaggatg aagctgattc ttctcttaca aggggcccag agccttcttg ggagttcagc 6241 tccaagggat gagccccagg tgtctgccaa gtccccctct gtccaggcct gggacggctc 6301 tgggatcgag gggtcagagg cgctgagccc agggagagac acctgcgccc agagctatga 6361 caaagggtgg agggatgaca aggcagccag gagcgggcgc ctgcggggtg gcacagaggg 6421 gcagggcccg aggacaggtg tcctgatggg agtgtgagaa agggtcccct gtgcggcagc 6481 caggagggta ggggggttgt tcactggggc cctgtggggg cagctccttc ctgagctgcc 6541 gttccctccc cggcagccga tgccactgtc catcaagaca tcgccctctt cccatcacta 6601 atccagttag cgcctggcct ggggatgagt gacacagcgt ctctgtctgt ctgctcgcca 6661 cagagtgggg agcaggcgag caccttccca gcccccactc ctcccccacc accactgctt 6721 ctgactgggc tgcccccatc gggaagggcg tgcaatgccc gcaggcacct cggctagcat 6781 ctgccccagc aggcacacag taggcgctca aaaacgtgct ctcatcccct gcctctgtgt 6841 gccatcagcg ctgcccgact gtgggaccag ctgtgggtgg aggtccccgg gtctcagcag 6901 gtggaggagg catgggtgcc ccttgtcccc acagtccccg cggttcattg ggcgcaggca 6961 gagcctcatc gaggacgccc gcaaggagcg ggaggcggcg gtggcagcag cggccgctgc 7021 agtcccctcg gagcccgggg accccctgga ggctgtggcc tttgaggaga aggaggggaa 7081 ggccgtgcta aacctgctct tctccccgag ggccaccaag ccctcggcgc tgtcccgagc 7141 tgtgaaggtg tttgaggtga gctggtggcc ttcgtgtccc tggggcaagt tcacctgtgg 7201 gtggggctgt gtgggctgag ttcctgaccc ctctatagca gaggtgcagc tgcccaggcc 7261 cccgaggccg gcacaggatg cagcagggga gtctcaggcc tcagctcagc ccccatggca 7321 tctagccaca cccccgtgtt tttgagggat cctgagccca cccctagggc tgaggctacc 7381 aagccccact gtgcctcttg ccttgcccat cccctggatc cccctcaccc accatttccc 7441 acgtgggggg ctcccagcag ggcagcacaa gaggcagggg cagggcagtg tgccctctcc 7501 cacccaccca gcacagtggc tcaggtgacc actgattgca ttagtcactc cggccccact 7561 gtgccccggg aggcaggtga cccagctccc ggaagaagct cccaaatgac attaaagcca 7621 gactccccgc cccccagctc ccagagccag ttttgtggcc cgagggccac tgcgacccac 7681 cgcccttgtt gctaggcaac aggaggtggg ggtggagcgg acccttctgg ccagtgtcct 7741 ggacgctcag gggccagtga gactcagggc ccatcctaca aacctggatg aggccaccag 7801 ggttgggggc accttctgac cagtggctga ggagccggac tgtgtggcat ggccttggga 7861 cacacacacc gagccgccca gaaccaggtt aagcctcaag cggtgacaac tcctggttag 7921 gcacgtaaca caaaatccaa cttgccagtg gcaaaccctg gcctggtggc cgacagctga 7981 cctgagcctg gaagaacggg atctgtgtgc tgctagcaca aaagtcaagg gcagggcctg 8041 gccagccagc cagatgtgcc tcctccccgc ccaccccacc ctctctctcc atctctgtct 8101 ctttctcctt ctctctctct tcctgctttt gctccctaag acgtttgaag ccaaaatcca 8161 ccatctagag acccggcccg cccagaggcc gcgagctggg ggcccccacc tggagtactt 8221 cgtgcgcctc gaggtgcgcc gaggggacct ggccgccctg ctcagtggtg tgcgccaggt 8281 gtcagaggac gtgcgcagcc ccgcggggcc caagggtgag gcggttttct gtccttgagg 8341 gccaccaaat gaccttgaga ggctggggtg caggggctcc tgcaggggga ccctacagtg 8401 accacgtggt ggtggcctgg ttccctctct gcgggctcca ctccgcaccc cgttttgcta 8461 cacatccgtg tccgggcctg gggccactcc aggatccccc cgcagctctc acagccccgg 8521 ctgcctctgc cccccggaag tcttgtaggg gaggctgctt caaggtgggt gacacagccc 8581 cacggctccg agctcaccaa gatctcttcc tccatcaccc ataaagtccc ctggttccca 8641 agaaaagtgt cagagctgga caagtgtcat cacctggtca ccaagttcga ccctgacctg 8701 gacttggacc acccggtgag tggtgcgccc ctcactcagg cctcctgccc ctgatcacat 8761 cccctaccct tagcccaacc ctggacagga gtctgtcggc tccaggagcc tccgtggcct 8821 gtgcccccac cccagcacag cctcctgacc cgtgcatccc ctctgccctc agggcttctc 8881 ggaccaggtg taccgccagc gcaggaagct gattgctgag atcgccttcc agtacaggca 8941 gtgaggggcc cctgcgctcg ggacccagac tccgtcctgc aggctgacgc tggacctggg 9001 gggtgggagg gaaggacaaa ggggaggacc catcttgtca ccagcatcag tgcctcctgc 9061 caggcagctc tgctccaggg ctttccatgt ccccaaatcc cagtggggaa actgaggccc 9121 aggggggcta gagcaacctg ccgaggccac atagccggct cacggcacag tcagctgggg 9181 tgcaccctcc tgtccatcct ccaacccaaa ggcctcgctg cactaggcgg gtgtggacct 9241 gtgcccagtg aagctccctc cctccctcct gcccttctca ctccccgagg ggacctgctg 9301 accactggcc ccctccccag cggcgacccg attccccgtg tggagtacac cgccgaggag 9361 attgccacct ggtgagacct ccgtgcagct aggggctggg gaggagcccg ggggatgcct 9421 cctggaatcc tggcgtgtga gggccgcctc cagggacctt ggcacaacag gagagactaa 9481 ggccgggaag aagagggact tgcagggctc agaatgttgg gttgggagga agaggctacc 9541 catcctgtcg ggccatcccc agtgtgctga gggaccgccc ctcatggccc cctatcccct 9601 gggattccct aaagccacca gcaaaagccc ctcccggggg cctgggtctt caggggtccc 9661 caagaggcct gcgttggtag gggctcaggc aggcagaggc acccacagtt caggaggggg 9721 gtttcgggca ctggggtggg gcattagagg gccctgagcc tggctgcccg caggaaggag 9781 gtctacacca cgctgaaggg cctctacgcc acgcacgcct gcggggagca cctggaggcc 9841 tttgctttgc tggagcgctt cagcggctac cgggaagaca atatccccca gctggaggac 9901 gtctcccgct tcctgaaggg tgtgcccaga cgggaggggc gcagagccgg ggggccgggg 9961 atggtcagcc aagcgcccca ccccagcgcg gctccagccc gtcccggctc ggcagtgacc10021 cgcgtggccc cttgcagagc gcacgggctt ccagctgcgg cctgtggccg gcctgctgtc10081 cgcccgggac ttcctggcca gcctggcctt ccgcgtgttc cagtgcaccc agtatatccg10141 ccacgcgtcc tcgcccatgc actcccctga gccgtgagtg cgcgccctgg ccgccagccc10201 gagggtgggg ggtgcgacgg gcggcccctc agcccccttc tccctcctac gcgcagggac10261 tgctgccacg agctgctggg gcacgtgccc atgctggccg accgcacctt cgcgcagttc10321 tcgcaggtac gccgcggcct cggagggagc cggggtcacc caggggctgg cttggcgccg10381 ggggcgggcg gggatcgatg tgcgggtggg tgaagtgtgc tgcctgctcc cgggccccgc10441 caaggaggct cggcgccccg agggtcgcgc ggcatagggc ggggctggag cggagcctcc10501 cacggcctgt gctgccacct gccggctacc tgggaacggc gcccacgggc ttaggaatgt10561 ggtcaaggag ggctgcctgg aggaggaggc ccggtggagg tgcggatcct gggcggccag10621 ggaaggtctc tgccgccagg gaagtgtccc agagacccct ggaggggctg ctgacacccc10681 cggtgccccc acctcgagca tgacccaggg ctgcctctcc ccatccttca tcctccctgc10741 tccacaggac attggcctgg cgtccctggg ggcctcggat gaggaaattg agaagctgtc10801 cacggtgggt tgacccctcc ctgcagggcc tggggtgtgg gtttgggggt ctgaatccag10861 gcctcaccct cttgccgtcc aggctgaggc ctctccttcc acccacgaat tgtgaccctc10921 accctggcct gcctgcatcc tggcctggcc tccctggggg tggtatcctg gtcacgggtg10981 accaggggct gcccggtggg cggcagctgt ctctgggctg atgctgcccg gcttccccgc11041 agctgtactg gttcacggtg gagttcgggc tgtgtaagca gaacggggag gtgaaggcct11101 atggtgccgg gctgctgtcc tcctacgggg agctcctggt gagagtctct ccttgctgca11161 gcccccagca gaggggcagg gctgggggac ggtgcaggga ggggacaggc tcccagtggg11221 aggaaactga ggcctggacc tccaggactc aggctctgtt tgggagaagg cttgtctctg11281 cccagtcctc accccacatt atcccaggcc tccgaaggcc cggcggggga gatgggggtg11341 actctaccca aggaacccac ccagcgtcag gccacggtgc cccagttccc tcggggacct11401 gggtgcagtg gagtcagtga tgccattggc ctcctgccag cactgcctgt ctgaggagcc11461 tgagattcgg gccttcgacc ctgaggctgc ggccgtgcag ccctaccaag accagacgta11521 ccagtcagtc tacttcgtgt ctgagagctt cagtgacgcc aaggacaagc tcaggtgggc11581 taggctgcta gggcaagccc cccatggtgc ccccaaactg ggccagccag gccttccttc11641 tggccttgag cagggctgga cctgtgagcc caggtcacag atgagaaaac cgacccctgg11701 ttgcagcagc ccccacacag cagggacacc atccgtgaga aggaccccag cgtctgggga11761 ggggcagacc tacaggactg ggggctgctg ggtggccggg tcaaggccag tcttggaggt11821 gctgacagag cctgagcttt gtgaggacgt cctgtggaac ctgtcccggc cccctgccct11881 gggatgggga gaagtcaggg ggatagacag agtcaaggtg ggggacaggg cgggagtggg11941 gtccccaggg ctgggggcct ttggtgcagt gaccagagtg tcaggagagg ggagcaaagc12001 cctctagcct catcctcata aaaggtctca tcattttccc tccagcctct tatgcactgg12061 ggaaactgag gccaggggct atgtgtccag cggacagggg tgctgaattc cacccacagg12121 cttagggata tggtcaagga aagcttcctg gaggaggccc agtggaggtt cagggaggga12181 tggggtgccc ggcagtctct agtggaaaag gcgcctagcc tatctccccc atgaaccccc12241 tcacccagcc ctggaagagg cctcagtgtc ccgcctgtga ccagttggct cagaaaagcc12301 ctgggagctc tgagccactg tgaaggtgga aacgcggccc ctggcctccc ctctcctgga12361 ggctgcagac tctgcccgcc agttgacgag ggctctgccg ctctcctccc caggagctat12421 gcctcacgca tccagcgccc cttctccgtg aagttcgacc cgtacacgct ggccatcgac12481 gtgctggaca gcccccaggc cgtgcggcgc tccctggagg gtgtccagga tgagctggac12541 acccttgccc atgcgctgag tgccattggc taggtgcacg gcgtccctga gggcccttcc12601 caacctcccc tggtcctgca ctgtcccgga gctcaggccc tggtgagggg ctgggtcccg12661 ggtgcccccc atgccctccc tgctgccagg ctcccactgc ccctgcacct gcttctcagc12721 gcaacagctg tgtgtgcccg tggtgaggtt gtgctgcctg tggtgaggtc ctgtcctggc12781 tcccagggtc ctgggggctg ctgcactgcc ctccgccctt ccctgacact gtctgctgcc12841 ccaatcaccg tcacaataaa agaaactgtg gtctctacac ctgcctggcc ccacatctgt12901 gccacagaga cagaccctgg gatcctcaga ctcccacacc cccaccccag cctcactcag12961 aggtttcgcc ctggcctcct tcctcctctg ggagatggct ggccgccctg gccaggcagc13021 tggcccctcc gggcctggtt tccccgctca ccctgaggcc ccgcccagct ctgagcccca13081 agcagctcca gaggctcggg caccctggcc gagctgcccc atctccgtgg ggtgccctcc13141 caaggtgggg agccacgtga cagtgggagg gcctctctca ggcctggcag ggagcagggg13201 tcacaaactg tgctggctgg gggtggtctc agaggtgggc ctgcaggcct aaccctccct13261 gctgacaggg ctcccagccc ttgagagaaa cagggatgga ggaacagctg ccctgatgcc13321 ctcacccacc cggagcaggc cctgcgaacc aaggggaacc tcagtgtggc ccccagcatg13381 tgtgctgatg gggagggtct ggctgagctg gtgcccaggc agatggtctg ggcctgtctc13441 cccagcgagg caggatgggg gctggatttc agactctgta agatgcccct ggcttactcg13501 aggggcctgg acattgccct ccagagagag cacccaacac cctccaggct tgaccggcca13561 gggtgtcccc ttcctacctt ggagagagca gccccagggc atcctgcagg gggtgctggg13621 acaccagctg gccttcaagg tctctgcctc cctccagcca ccccactaca cgctgctggg13681 atcctggatc tcagctcccc ggccgacaac actggcaaac tcctactcat ccacgaaggc13741 cctcctgggc atggtggtcc ttcccagcct ggcagtctgt tcctcacaca ccttgttagt13801 gcccagcccc tgaggttgca gctgggggtg tctctgaagg gctgtgagcc cccaggaagc13861 cctggggaag tgcctgcctt gcctcccccc ggccctgcca gcgcctggct ctgccctcct13921 acctgggctc cccccatcca gcctccctcc ctacacactc ctctcaagga ggcacccatg13981 tcctctccag ctgccgggcc tcagagcact gtggcgtcct ggggcagcca ccgcatgtcc14041 tgctgtggca tggctcaggg tggaaagggc ggaagggagg ggtcctgcag atagctggtg14101 cccactacca aacccgctcg gggcaggaga gccaaaggct gggtgtgtgc agagcggccc14161 cgagaggttc cgaggctgag gccagggtgg gacataggga tgcgaggggc cggggcacag14221 gatactccaa cctgcctgcc cccatggtct catcctcctg cttctgggac ctcctgatcc14281 tgcccctggt gctaagaggc aggtaggggc tgcaggcagc agggctcgga gcccatgccc14341 cctcaccatg ggtcaggctg gacctccagg tgcctgttct ggggagctgg gagggccgga14401 ggggtgtacc ccaggggctc agcccagatg acactatggg ggtgatggtg tcatgggacc14461 tggccaggag aggggagatg ggctcccaga agaggagtgg gggctgagag ggtgcctggg14521 gggccaggac ggagctgggc cagtgcacag cttcccacac ctgcccaccc ccagagtcct14581 gccgccaccc ccagatcaca cggaagatga ggtccgagtg gcctgctgag gacttgctgc14641 ttgtccccag gtccccaggt catgccctcc ttctgccacc ctggggagct gagggcctca14701 gctggggctg ctgtcctaag gcagggtggg aactaggcag ccagcaggga ggggacccct14761 ccctcactcc cactctccca cccccaccac cttggcccat ccatggcggc atcttgggcc14821 atccgggact ggggacaggg gtcctgggga caggggtgtg gggacagggg tcctggg

By “LMX1A polypeptide” (or LIM homeobox transcription factor 1-alpha) ismeant a polypeptide or fragment thereof having at least about 85% aminoacid identity to NCBI Accession No. Q8TE12.

1 mldglkmeen fqsaidtsas fssllgrays pksvcegcqr vildrfllrl ndsfwheqcv 61qcasckeple ttcfyrdkkl yckydyeklf avkcggcfea iapnefvmra qksvyhlscf 121cccvcerqlq kgdefvlkeg qllckgdyek erellslvsp aasdsgksdd eeslcksahg 181agkgtaeegk dhkrpkrprt ilttqqrraf kasfevsskp crkvretlaa etglsvrvvq 241vwfqnqrakm kklarrqqqq qqdqqntqrl ssaqtngggs agmegimnpy talptpqq11 301aieqsvyssd pfrqgltppq mpgdhmhpyg aeplfhdlds ddtslsnlgd cflatseagp 361lqsrvgnpid hlysmqnsyf ts

By “LMX1A nucleic acid molecule” (or LIM homeobox transcription factor1-alpha) is meant a polynucleotide encoding an LMX1A polypeptide. Anexemplary LMX1A nucleic acid molecule is provided at NCBI Accession No.AH011517.

1 gtataggttg gggcggagtc ggattcggga tggaaaacct ggggcaaggg atgtaggtgg 61gggtgagggg ggcaggagaa ggagaaacgc agttgggggg cggaggccta agtacataac 121gtgttgactt caagtgaaat cagatcagcc agagcagttc gctgtgactg atctctcctc 181ccaccctaca ttctcttggc tggaccctat cctcctggct gattctggtc gccctggaca 241ctccctcagt tctttcccag gagtgcggtg gctgctggcg ccgagtccca gcgggcacgg 301acgtcagacg catcgtttct tctcctctac aggtcctccc ggcccggccc gaacatgctg 361gacggcctaa agatggagga gaacttccaa agcgcgatcg acacctcggc ctccttctcc 421tcgctgctgg gtgagtgttc aggccgtgcg tcctgggcgc actctctttc cgcttggcgc 481tgagctctgg agccccgctc tctgggacct ggtccgcgat agggaagcta gcgcccctct 541tcatacacta aattgagccc catcactatc tgtccgtcag tgcttgtggg tcgtccctac 601ccaaataaat ccaacaagcc gccccaggcc tcacgcactg ggcaccgaat tccccaaagc 661cgcgaggggc gggcgagctt gttcgtaggc gtctgagtgg caagtgatta aaaataccca 721gggctggatt tttaatctcg gagctgatcg acgtctcata aatgccgccc tcttctcgcg 781gcctagaggc aatagcatcc gagacccgag gcctggagcg cccaagttcg aggaggcttc 841tctcccccac caactccagc cccaatttca gccatgggca aggccgagag agacttttct 901nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 961nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn gggtcccggc caggtttggc 1021atggtctacc tgcccgggct gctcacccgc caacgtctgt tgtggctaca ggcagagcgg 1081tgagccccaa gtctgtctgc gagggctgtc agcgggtcat cttggacagg tttctgctgc 1141ggctcaacga cagcttctgg catgagcagt gcgtgcagtg cgcctcctgc aaagagcccc 1201tggagaccac ctgcttctac cgggacaaga agctgtactg caagtatgac tacgagaagt 1261aagtggccgc acccccgcag cgctccccgc gcactggcat nnnnnnnnnn nnnnnnnnnn 1321nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1381nnnnnnnnnn nnnnnnnnnn atcccagttc ttgaagttcc ttttgctgtt gacttcaggg 1441gagacccagg accaagccag attttactca tggtgcatgt acttcctttc tccctgctgc 1501caggctgttt gctgttaaat gtgggggctg cttcgaggcc atcgctccca atgagtttgt 1561tatgcgggcc cagaagagtg tataccacct gagctgcttc tgctgctgtg tctgcgagcg 1621acagcttcag aagggtgatg agtttgtcct gaaggagggg cagctgctct gcaaagggga 1681ctatgagaag gagcgggagc tgctcagcct ggtgagccca gcagcctcag actcaggtga 1741gtgccaggtg gtgggcaggg ctgcggtggg gtgggtagag tggagttggg tggctgtctg 1801cattgtttct tccctagatg nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1861nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1921catacagctc caggaactgg ctttcaggga ctcacaacat tgtcttttgc ttctttcagg 1981taaaagtgat gatgaagaaa gtctctgcaa gtcagcccat ggggcaggga aaggaactgc 2041tgaggaaggc aaggaccata agcgccccaa acgtccgaga accatcttga caactcaaca 2101gaggcgagca ttcaaggcct catttgaagt atcctccaag ccctgcagga aggtatagga 2161gggagcaggg aggaaaagga gctgggcccc acttctctgt gtgcactcag acccctctgg 2221gatctcagtg ggcattgggg gtcacagtgg tgaggaaggc tgttcagaca gagcctgcac 2281aggcggctca agcctgttgg agactccaga gatcactaag ctgtggccag ggtgtgatag 2341actctcctga agctttcatg catgcacacc aactccaaat ggcccctgtc acacctttca 2401tttcatagag cacaatggga acagtaataa tgataggtgt ccattgtggt gtagacccag 2461atgctgtaaa gcaaagagta taaaaacaca gtggcttgca gtactctttt ttgagtctgg 2521ctttttccac ttggtgtggt ggtttgggga ttcattcatt cctatttcag cattccactg 2581tataggtgtg ccatgattgg tttgtccatg cacctgttga tgggtgtttg gggttgtttc 2641tagtttggga ctgtttcaaa taggactgct atggacattc atgtaaaaaa aaatacagtg 2701gtttaatgag acaggagttt attctcttct gtcacagtcc agaggtgagc aaggcaaggc 2761tggtgggtgg ctctgttatc catctcctgt gtccaagcga ctgctccagt tgtcaccatg 2821tttccagtca ccaggtagag aaagaggaaa tggagggcaa gcgccctgct ttttaaggat 2881nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2941nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn atgcatatgc atggcttata 3001gctaaagcac aacaatagac taaagtctaa accacttgaa ggcctaattt ccagagcaag 3061agaaatccag aaacacctct tgggaatgca catgtaaatt aataattatt attttgtttc 3121tttacctggt gaaggacttt ctttctacct gaagggaagc aatgttctcg tgtttgtgtg 3181tatgctcaac attaaaaact attcagctcc taaagcagat acagtctttt ggcctcctca 3241agtattatat aggagatgtt ctacctccta ccctgagatg ccagtgtgtc tacatttctc 3301gttcaatttt tccaaggtga gagagactct ggctgcagag acagggctga gtgtccgtgt 3361cgtccaggtg tggttccaaa accagagagc gaaggtaacc tgcttcttac ttttatctgt 3421ccccatgttg ctggtttcct gaaataatca cagtaggaca nnnnnnnnnn nnnnnnnnnn 3481nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3541nnnnnnnnnn nnnnnnnnnn agccctctcc cggggaaggt gtcacttcca ggccccccct 3601tactttgtga acatgctgca ggccacctga cttctaatcc tatggtcctc tccttatcag 3661atgaagaagc tggccaggcg acagcagcag cagcagcaag atcagcagaa cacccagagg 3721ctgagctctg gtaagctggt gcctcctccc aggcagttct ggctggaatc caggctgttc 3781ctaccagagg cctcccacta cccagctctt tggatgacat atctggactc agtgaagcct 3841agaccacacc cactggagaa ataaggcctt caagggaaga ctgagccacg aggaacttgt 3901gagagggttg agggctcctg agctgcaggc ttagaactgc tgattgggga tggcactgac 3961cttatccaca gcgtccaggc ctggatccca ccacagcgtc agggactgct tgcagagtca 4021cagatacgtt cagtttctca tcttgcttag ttctccttcc aggctaattg atttaataga 4081agacacctcg gtgacttggc tctttccaaa ataacataaa gtagtaaaaa taatgatagt 4141aaaataacaa tgccttcctt tgttgaacac tcttatagat tggtgttctc atacatgctg 4201acttgacttt tacaacaccc attcctggag gcgagtggag aagttgttat tatccctatg 4261tcacagatga gcaaacaaag gctctgcaag attgaatgtg gccctagatc ggtaagggca 4321gggggctggg actagaactc taactgtgtt ccacaggcca tgggccttct catctctacc 4381cagatgtgct tttgaaaaag nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4441nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4501cacgttgaga atgacctggc ttcttctttg ttccacagct cagacaaacg gtggtgggag 4561tgctgggatg gaaggaatca tgaaccccta cacggctctg cccaccccac agcagctcct 4621ggccatcgag cagagtgtct acagctcaga tcccttccga cagggtctca ccccacccca 4681gatgcctgga gaccacatgc acccttatgg taagagggac ttaagcccct cgggccctct 4741cataacttgt gtgggtttct cattccctcc taaacacatc taggcagttc ccagatgctc 4801nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4861nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn aaatgagtca cacttcaag 4921accctcatgc cagtgtttca tctccatttc aggtgccgag ccccttttcc atgacctgga 4981tagcgacgac acctccctca gtaacctggg tgattgtttc ctagcaacct cagaagctgg 5041gcctctgcag tccagagtgg gaaaccccat tgaccatctg tactccatgc agaattctta 5101cttcacatct tgagtcttcc cctagagttc tgtgactagg ctcccatatg gaacaaccat 5161attctttgag gggtcactgg ctttaggaca gggaggccag ggaagaggtg ggttggggag 5221ggagttttgt tggggatgct gttgtataat gatatggtgt agctcagcat ttccaaagac 5281tgaatacatt atggattgca tagtttaatg

By “FOXO1 polypeptide” (or Forkhead box protein 01) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. Q12778.

1 maeapqvvei dpdfeplprp rsctwplprp efsqsnsats spapsgsaaa npdaaaglps 61asaaaysadf msnlsllees edfpqapgsv aaavaaaaaa aatgglcgdf qgpeagclhp 121appqppppgp lsqhppvppa aagplagqpr kssssrrnaw gnlsyadlit kaiessaekr 181ltlsqiyewm vksvpyfkdk gdsnssagwk nsirhnlslh skfirvqneg tgksswwmln 241peggksgksp rrraasmdnn skfaksrsra akkkaslqsg qegagdspgs qfskwpaspg 301shsnddfdnw stfrprtssn astisgrlsp imteqddlge gdvhsmvypp saakmastlp 361slseisnpen menlldnlnl lssptsltvs tqsspgtmmq qtpcysfapp ntslnspspn 421yqkytygqss msplpqmpiq tlqdnkssyg gmsqyncapg llkelltsds pphndimtpv 481dpgvaqpnsr vlgqnvmmgp nsvmstygsq ashnkmmnps shthpghaqq tsavngrplp 541htvstmphts gmnrltqvkt pvqvplphpm qmsalggyss vsscngygrm gllhqeklps 601dldgmfierl dcdmesiirn dlmdgdtldf nfdnvlpnqs fphsvkttth swvsg

By “FOXO1 nucleic acid molecule” (or Forkhead box protein 01) is meant apolynucleotide (e.g., mRNA) encoding an FOXO1 polypeptide. An exemplaryFOXO1 nucleic acid molecule is provided at NCBI Accession No. NM_002015.

1 gcagccgcca cattcaacag gcagcagcgc agcgggcgcg ccgctgggga gagcaagcgg 61cccgcggcgt ccgtccgtcc ttccgtccgc ggccctgtca gctggagcgc ggcgcaggct 121ctgccccggc ccggcggctc tggccggccg tccagtccgt gcggcggacc ccgaggagcc 181tcgatgtgga tggccccgcg aagttaagtt ctgggctcgc gcttccactc cgccgcgcct 241tcctcccagt ttccgtccgc tcgccgcacc ggcttcgttc ccccaaatct cggaccgtcc 301cttcgcgccc cctccccgtc cgcccccagt gctgcgttct ccccctcttg gctctcctgc 361ggctggggga ggggcggggg tcaccatggc cgaggcgcct caggtggtgg agatcgaccc 421ggacttcgag ccgctgcccc ggccgcgctc gtgcacctgg ccgctgccca ggccggagtt 481tagccagtcc aactcggcca cctccagccc ggcgccgtcg ggcagcgcgg ctgccaaccc 541cgacgccgcg gcgggcctgc cctcggcctc ggctgccgct gtcagcgccg acttcatgag 601caacctgagc ttgctggagg agagcgagga cttcccgcag gcgcccggct ccgtggcggc 661ggcggtggcg gcggcggccg ccgcggccgc caccgggggg ctgtgcgggg acttccaggg 721cccggaggcg ggctgcctgc acccagcgcc accgcagccc ccgccgcccg ggccgctgtc 781gcagcacccg ccggtgcccc ccgccgccgc tgggccgctc gcggggcagc cgcgcaagag 841cagctcgtcc cgccgcaacg cgtggggcaa cctgtcctac gccgacctca tcaccaaggc 901catcgagagc tcggcggaga agcggctcac gctgtcgcag atctacgagt ggatggtcaa 961gagcgtgccc tacttcaagg ataagggtga cagcaacagc tcggcgggct ggaagaattc 1021aattcgtcat aatctgtccc tacacagcaa gttcattcgt gtgcagaatg aaggaactgg 1081aaaaagttct tggtggatgc tcaatccaga gggtggcaag agcgggaaat ctcctaggag 1141aagagctgca tccatggaca acaacagtaa atttgctaag agccgaagcc gagctgccaa 1201gaagaaagca tctctccagt ctggccagga gggtgctggg gacagccctg gatcacagtt 1261ttccaaatgg cctgcaagcc ctggctctca cagcaatgat gactttgata actggagtac 1321atttcgccct cgaactagct caaatgctag tactattagt gggagactct cacccattat 1381gaccgaacag gatgatcttg gagaagggga tgtgcattct atggtgtacc cgccatctgc 1441cgcaaagatg gcctctactt tacccagtct gtctgagata agcaatcccg aaaacatgga 1501aaatcttttg gataatctca accttctctc atcaccaaca tcattaactg tttcgaccca 1561gtcctcacct ggcaccatga tgcagcagac gccgtgctac tcgtttgcgc caccaaacac 1621cagtttgaat tcacccagcc caaactacca aaaatataca tatggccaat ccagcatgag 1681ccctttgccc cagatgccta tacaaacact tcaggacaat aagtcgagtt atggaggtat 1741gagtcagtat aactgtgcgc ctggactctt gaaggagttg ctgacttctg actctcctcc 1801ccataatgac attatgacac cagttgatcc tggggtagcc cagcccaaca gccgggttct 1861gggccagaac gtcatgatgg gccctaattc ggtcatgtca acctatggca gccaggcatc 1921tcataacaaa atgatgaatc ccagctccca tacccaccct ggacatgctc agcagacatc 1981tgcagttaac gggcgtcccc tgccccacac ggtaagcacc atgccccaca cctcgggtat 2041gaaccgcctg acccaagtga agacacctgt acaagtgcct ctgccccacc ccatgcagat 2101gagtgccctg gggggctact cctccgtgag cagctgcaat ggctatggca gaatgggcct 2161tctccaccag gagaagctcc caagtgactt ggatggcatg ttcattgagc gcttagactg 2221tgacatggaa tccatcattc ggaatgacct catggatgga gatacattgg attttaactt 2281tgacaatgtg ttgcccaacc aaagcttccc acacagtgtc aagacaacga cacatagctg 2341ggtgtcaggc tgagggttag tgagcaggtt acacttaaaa gtacttcaga ttgtctgaca 2401gcaggaactg agagaagcag tccaaagatg tctttcacca actccctttt agttttcttg 2461gttaaaaaaa aaaacaaaaa aaaaaaccct ccttttttcc tttcgtcaga cttggcagca 2521aagacatttt tcctgtacag gatgtttgcc caatgtgtgc aggttatgtg ctgctgtaga 2581taaggactgt gccattggaa atttcattac aatgaagtgc caaactcact acaccatata 2641attgcagaaa agattttcag atcctggtgt gctttcaagt tttgtatata agcagtagat 2701acagattgta tttgtgtgtg tttttggttt ttctaaatat ccaattggtc caaggaaagt 2761ttatactctt tttgtaatac tgtgatgggc ctcatgtctt gataagttaa acttttgttt 2821gtactacctg ttttctgcgg aactgacgga tcacaaagaa ctgaatctcc attctgcatc 2881tccattgaac agccttggac ctgttcacgt tgccacagaa ttcacatgag aaccaagtag 2941cctgttatca atctgctaaa ttaatggact tgttaaactt ttggaaaaaa aaagattaaa 3001tgccagcttt gtacaggtct tttctatttt tttttgttta ttttgttatt tgcaaatttg 3061tacaaacatt taaatggttc taatttccag ataaatgatt tttgatgtta ttgttgggac 3121ttaagaacat ttttggaata gatattgaac tgtaataatg ttttcttaaa actagagtct 3181actttgttac atagtcagct tgtaaatttt gtggaaccac aggtatttgg ggcagcattc 3241ataattttca ttttgtattc taactggatt agtactaatt ttatacatgc ttaactggtt 3301tgtacacttt gggatgctac ttagtgatgt ttctgactaa tcttaaatca ttgtaattag 3361tacttgcata ttcaacgttt caggccctgg ttgggcagga aagtgatgta tagttatgga 3421cactttgcgt ttcttattta ggataactta atatgttttt atgtatgtat tttaaagaaa 3481tttcatctgc ttctactgaa ctatgcgtac tgcatagcat caagtcttct ctagagacct 3541ctgtagtcct gggaggcctc ataatgtttg tagatcagaa aagggagatc tgcatctaaa 3601gcaatggtcc tttgtcaaac gagggatttt gatccacttc accattttga gttgagcttt 3661agcaaaagtt tcccctcata attctttgct cttgtttcag tccaggtgga ggttggtttt 3721gtagttctgc cttgaggaat tatgtcaaca ctcatacttc atctcattct cccttctgcc 3781ctgcagatta gattacttag cacactgtgg aagtttaagt ggaaggaggg aatttaaaaa 3841tgggacttga gtggtttgta gaatttgtgt tcataagttc agatgggtag caaatggaat 3901agaacttact taaaaattgg ggagatttat ttgaaaacca gctgtaagtt gtgcattgag 3961attatgttaa aagccttggc ttaagaattt gaaaatttct ttagcctgta gcaacctaaa 4021ctgtaattcc tatcattatg ttttattact ttccaattac ctgtaactga cagaccaaat 4081taattggctt tgtgtcctat ttagtccatc agtattttca agtcatgtgg aaagcccaaa 4141gtcatcacaa tgaagagaac aggtgcacag cactgttcct cttgtgttct tgagaaggat 4201ctaatttttc tgtatatagc ccacatcaca cttgctttgt cttgtatgtt aattgcatct 4261tcattggctt ggtatttcct aaatgtttaa caagaacaca agtgttcctg ataagatttc 4321ctacagtaag ccagctctat tgtaagcttc ccactgtgat gatcattttt ttgaagattc 4381attgaacagc caccactcta tcatcctcat tttggggcag tccaagacat agctggtttt 4441agaaacccaa gttcctctaa gcacagcctc ccgggtatgt aactgaactt ggtgccaaag 4501tacttgtgta ctaatttcta ttactacgta ctgtcacttt cctcccgtgc cattactgca 4561tcataataca aggaacctca gagcccccat ttgttcatta aagaggcaac tacagccaaa 4621atcactgtta aaatcttact acttcatgga gtagctctta ggaaaatata tcttcctcct 4681gagtctgggt aattatacct ctcccaagcc cccattgtgt gttgaaatcc tgtcatgaat 4741ccttggtagc tctctgagaa cagtgaagtc cagggaaagg catctggtct gtctggaaag 4801caaacattat gtggcctctg gtagtttttt tcctgtaaga atactgactt tctggagtaa 4861tgagtatata tcagttattg tacatgattg ctttgtgaaa tgtgcaaatg atatcaccta 4921tgcagccttg tttgatttat tttctctggt ttgtactgtt attaaaagca tattgtatta 4981tagagctatt cagatatttt aaatataaag atgtattgtt tccgtaatat agacgtatgg 5041aatatattta ggtaatagat gtattacttg gaaagttctg ctttgacaaa ctgacaaagt 5101ctaaatgagc acatgtatcc cagtgagcag taaatcaatg gaacatccca agaagaggat 5161aaggatgctt aaaatggaaa tcattctcca acgatataca aattggactt gttcaactgc 5221tggatatatg ctaccaataa ccccagcccc aacttaaaat tcttacattc aagctcctaa 5281gagttcttaa tttataacta attttaaaag agaagtttct tttctggttt tagtttggga 5341ataatcattc attaaaaaaa atgtattgtg gtttatgcga acagaccaac ctggcattac 5401agttggcctc tccttgaggt gggcacagcc tggcagtgtg gccaggggtg gccatgtaag 5461tcccatcagg acgtagtcat gcctcctgca tttcgctacc cgagtttagt aacagtgcag 5521attccacgtt cttgttccga tactctgaga agtgcctgat gttgatgtac ttacagacac 5581aagaacaatc tttgctataa ttgtataaag ccataaatgt acataaatta tgtttaaatg 5641gcttggtgtc tttcttttct aattatgcag aataagctct ttattaggaa ttttttgtga 5701agctattaaa tacttgagtt aagtcttgtc agccacaa

By “FOXA2 polypeptide” (or Forkhead box protein A2) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. Q9Y261.

1 mlgavkmegh epsdwssyya epegyssysn mnaglgmngm ntymsmsaaa mgsgsgnmsa 61gsmnmssyvg agmspslagm spgagamagm ggsagaagva gmgphlspsl splggqaaga 121mgglapyanm nsmspmygqa glsrardpkt yrrsythakp pysyislitm aiqqspnkml 181tlseiyqwim dlfpfyrqnq qrwqnsirhs lsfndcflkv prspdkpgkg sfwdhpdsg 241nmfengcylr rqkrfkcekq lalkeaagaa gsgkkaaaga qasqaqlgea agpasetpag 301tesphssasp cqehkrgglg elkgtpaaal sppepapspg qqqqaaahll gpphhpglpp 361eahlkpehhy afnhpfsinn lmsseqqhhh shhhhqphkm dlkayeqvmh ypgygspmpg 421slamgpvtnk tgldasplaa dtsyyqgvys rpimnss

By “FOXA2 nucleic acid molecule” (or Forkhead box protein A2) is meant apolynucleotide (e.g., mRNA) encoding an FOXA2 polypeptide. An exemplaryFOXA2 nucleic acid molecule is provided at NCBI Accession No. NM_021784.

1 cccgcccact tccaactacc gcctccggcc tgcccaggga gagagaggga gtggagccca 61gggagaggga gcgcgagaga gggagggagg aggggacggt gctttggctg actttttttt 121aaaagagggt gggggtgggg ggtgattgct ggtcgtttgt tgtggctgtt aaattttaaa 181ctgccatgca ctcggcttcc agtatgctgg gagcggtgaa gatggaaggg cacgagccgt 241ccgactggag cagctactat gcagagcccg agggctactc ctccgtgagc aacatgaacg 301ccggcctggg gatgaacggc atgaacacgt acatgagcat gtcggcggcc gccatgggca 361gcggctcggg caacatgagc gcgggctcca tgaacatgtc gtcgtacgtg ggcgctggca 421tgagcccgtc cctggcgggg atgtcccccg gcgcgggcgc catggcgggc atgggcggct 481cggccggggc ggccggcgtg gcgggcatgg ggccgcactt gagtcccagc ctgagcccgc 541tcggggggca ggcggccggg gccatgggcg gcctggcccc ctacgccaac atgaactcca 601tgagccccat gtacgggcag gcgggcctga gccgcgcccg cgaccccaag acctacaggc 661gcagctacac gcacgcaaag ccgccctact cgtacatctc gctcatcacc atggccatcc 721agcagagccc caacaagatg ctgacgctga gcgagatcta ccagtggatc atggacctct 781tccccttcta ccggcagaac cagcagcgct ggcagaactc catccgccac tcgctctcct 841tcaacgactg tttcctgaag gtgccccgct cgcccgacaa gcccggcaag ggctccttct 901ggaccctgca ccctgactcg ggcaacatgt tcgagaacgg ctgctacctg cgccgccaga 961agcgcttcaa gtgcgagaag cagctggcgc tgaaggaggc cgcaggcgcc gccggcagcg 1021gcaagaaggc ggccgccgga gcccaggcct cacaggctca actcggggag gccgccgggc 1081cggcctccga gactccggcg ggcaccgagt cgcctcactc gagcgcctcc ccgtgccagg 1141agcacaagcg agggggcctg ggagagctga aggggacgcc ggctgcggcg ctgagccccc 1201cagagccggc gccctctccc gggcagcagc agcaggccgc ggcccacctg ctgggcccgc 1261cccaccaccc gggcctgccg cctgaggccc acctgaagcc ggaacaccac tacgccttca 1321accacccgtt ctccatcaac aacctcatgt cctcggagca gcagcaccac cacagccacc 1381accaccacca accccacaaa atggacctca aggcctacga acaggtgatg cactaccccg 1441gctacggttc ccccatgcct ggcagcttgg ccatgggccc ggtcacgaac aaaacgggcc 1501tggacgcctc gcccctggcc gcagatacct cctactacca gggggtgtac tcccggccca 1561ttatgaactc ctcttaagaa gacgacggct tcaggcccgg ctaactctgg caccccggat 1621cgaggacaag tgagagagca agtgggggtc gagactttgg ggagacggtg ttgcagagac 1681gcaagggaga agaaatccat aacaccccca ccccaacacc cccaagacag cagtcttctt 1741cacccgctgc agccgttccg tcccaaacag agggccacac agatacccca cgttctatat 1801aaggaggaaa acgggaaaga atataaagtt aaaaaaaagc ctccggtttc cactactgtg 1861tagactcctg cttcttcaag cacctgcaga ttctgatttt tttgttgttg ttgttctcct 1921ccattgctgt tgttgcaggg aagtcttact taaaaaaaaa aaaaaatttt gtgagtgact 1981cggtgtaaaa ccatgtagtt ttaacagaac cagagggttg tactattgtt taaaaacagg 2041aaaaaaaata atgtaagggt ctgttgtaaa tgaccaagaa aaagaaaaaa aaagcattcc 2101caatcttgac acggtgaaat ccaggtctcg ggtccgatta atttatggtt tctgcgtgct 2161ttatttatgg cttataaatg tgtattctgg ctgcaagggc cagagttcca caaatctata 2221ttaaagtgtt atacccggtt ttatcccttg aatcttttct tccagatttt tcattatt 2281acttggctta caaaatatac aggcttggaa attatttcaa gaaggaggga gggataccct 2341gtctggttgc aggttgtatt ttattttggc ccagggagtg ttgctgtttt cccaacattt 2401tattaataaa attttcagac ataaaaaa

By “FOXO4 polypeptide” (or Forkhead box protein 04) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. P98177.

1 mdpgnensat eaaaiidldp dfepqsrprs ctwplprpei anqpseppev epdlgekvht 61egrsepillp srlpepaggp qpgilgavtg prkggsrrna wgnqsyaeli sqaiesapek 121rltlaqiyew mvrtvpyfkd kgdsnssagw knsirhnlsl hskfikvhne atgksswwml 181npeggksgka prrraasmds sskllrgrsk apkkkpsvlp appegatpts pvghfakwsg 241spcsrnreea dmwttfrprs ssnassystr lsplrpesev laeeipasvs syaggvpptl 301neglelldgl nitsshslls rsglsgfslq hpgvtgplht yssslfspae gplsagegcf 361sssqaleall tsdtppppad vlmtqvdpil sqapt1111g glpsssklat gvglcpkple 421apgpsslvpt lsmiapppvm asapipkalg tpvltpptea asqdrmpqdl dldmymenle 481cdmdniisdl mdegegldfn fepdp

By “FOXO4 nucleic acid molecule” (or Forkhead box protein 04) is meant apolynucleotide (e.g., mRNA) encoding an FOXO4 polypeptide. An exemplaryFOXO4 nucleic acid molecule is provided at NCBI Accession No. NM 005938.

1 aaaaggggga gggaactgcg gctaaggaga cgttcggtga tgggagcgca atatatgagg 61ggatacagtg cctcaggttt aaaagagcag gaagctgagt gagaggttgc agaaaaagtg 121tcttcgctcg gcagaggtta caggtggcat ctcagaaaga gctttgaggc tacaggctgt 181agtcgggaag gggatcggag aactgtgtga agggacagct tagggactag cgtcctggga 241ctagggggaa gttcgcgact ttctgaagac tggcaggaat gtgcctcctg gccctcgatg 301cttcccccct gaggggaggc atcgtgaggg actgtggcag gcttcactga acgctgagcc 361ggggaggtcc aactccacgt atggatccgg ggaatgagaa ttcagccaca gaggctgccg 421cgatcataga cctagatccc gacttcgaac cccagagccg tccccgctcc tgcacctggc 481cccttccccg accagagatc gctaaccagc cgtccgagcc gcccgaggtg gagccagatc 541tgggggaaaa ggtacacacg gaggggcgct cagagccgat cctgttgccc tctcggctcc 601cagagccggc cgggggcccc cagcccggaa tcctgggggc tgtaacaggt cctcggaagg 661gaggctcccg ccggaatgcc tggggaaatc agtcatatgc agaactcatc agccaggcca 721ttgaaagcgc cccggagaag cgactgacac ttgcccagat ctacgagtgg atggtccgta 781ctgtacccta cttcaaggac aagggtgaca gcaacagctc agcaggatgg aagaactcga 841tccgccacaa cctgtccctg cacagcaagt tcatcaaggt tcacaacgag gccaccggca 901aaagctcttg gtggatgctg aaccctgagg gaggcaagag cggcaaagcc ccccgccgcc 961gggccgcctc catggatagc agcagcaagc tgctccgggg ccgcagtaaa gcccccaaga 1021agaaaccatc tgtgctgcca gctccacccg aaggtgccac tccaacgagc cctgtcggcc 1081actttgccaa gtggtcaggc agcccttgct ctcgaaaccg tgaagaagcc gatatgtgga 1141ccaccttccg tccacgaagc agttcaaatg ccagcagtgt cagcacccgg ctgtccccct 1201tgaggccaga gtctgaggtg ctggcggagg aaataccagc ttcagtcagc agttatgcag 1261ggggtgtccc tcccaccctc aatgaaggtc tagagctgtt agatgggctc aatctcacct 1321cttcccattc cctgctatct cggagtggtc tctctggctt ctctttgcag catcctgggg 1381ttaccggccc cttacacacc tacagcagct cccttttcag cccagcagag gggcccctgt 1441cagcaggaga agggtgcttc tccagctccc aggctctgga ggccctgctc acctctgata 1501cgccaccacc ccctgctgac gtcctcatga cccaggtaga tcccattctg tcccaggctc 1561cgactcttct gttgctgggg gggcttcctt cctccagtaa gctggccacg ggcgtcggcc 1621tgtgtcccaa gcccctagag gctccaggcc ccagcagtct ggttcccacc ctttctatga 1681tagcaccacc tccagtcatg gcaagtgccc ccatccccaa ggctctgggg actcctgtgc 1741tcacaccccc tactgaagct gcaagccaag acagaatgcc tcaggatcta gatcttgata 1801tgtatatgga gaacctggag tgtgacatgg ataacatcat cagtgacctc atggatgagg 1861gcgagggact ggacttcaac tttgagccag atccctgagt catgcctgga agctttgtcc 1921cctgcttcag atgtggagcc aggcgtgttc atatctactc tttacccttg agccctcccc 1981aggaatttgg gaccctgctt tagagctagg gtggggtctg gtcacacaca ggtgttgaag 2041aaattataaa gataaagctg ccccatctgg ggacgatatg gggagggaga tgggagggga 2101aaggggagag ggtttttctc actgtgccaa ttagggggta aggccccctc tcaggagcca 2161tcatcggctt tccccattcc tacccactta ggctttgtag caagatgagc aatgctgttg 2221gaaatgtgaa gtcaccagtg gccttacccc tgcctttggg agcaggattt ttttgtagag 2281agtcttatct gagctgagcc aggctagctg gagcctggga tttctatgca gtggcccctt 2341aggccagtga tgtgcggtgg gtgggctgtt taggggatct ggaagggcca aggtctgagc 2401actggagtgg ctcgccaggc caaatcaccc ttagaaggct gcagataaca gaaaggcttt 2461ttataaactt ttaaagaaat ataaacacaa atatagagat tttttaacca tggcagggtg 2521ctagtggtgg gcagaatgct tttttttctt tctgaaggct ttgtgatagt gacatgatac 2581aaacactaca gacaataaat attaggagac acagggaagt ggggagaggt ggggagtaat 2641agtaaacaca gggaagagct cccctacgga ccaggtatag agaaaggtct atgcagaaat 2701aggttagagt ttccctaaca aaaaagctaa cccaggtccc ctcattcctt caacttgtgc 2761ctgggagtgt gtggtgttag ggtgcagcca cactcttcta tgacccagca tgggttagtg 2821ctatggtggg agagtacatt gaaggcctgg aattagcttg gggccaggga agggactggg 2881aggggagaga agagaaggag ggaaggattt aggatggtaa agttaggtac agagacctcc 2941ctgttcaagg cccctgacag ctgtccctgc ccttcttccc cttccctgac tgcaggggtt 3001atgtggaagt gtgtgtggca gcaggcagcg gggaggggag gaacagggaa gggggagctg 3061gggagcttgg ctgagggtct gggaaatgag cagggatggg gggggatgtg gatcaggttt 3121actagcacct gccagggagg ccatctgggg ctccttctcc accccagccc ccaaagcagc 3181ccttccccca gtgccctttg catcgtcccc tcccccaccc ctgctgtggg ttcccatcat 3241ttcctgtgtc agcgcctggc ctacccagat tgtatcatgt gctagattgg agtggggaag 3301tgtgtcaaat caataaatga ataaattcaa taaatgccta taaccagcaa aaaaaaaaaa 3361aaaaa

By “CNP polypeptide” (or 2′,3′-cyclic-nucleotide 3′-phosphodiesterase)is meant a polypeptide or fragment thereof having at least about 85%amino acid identity to NCBI Accession No. P09543.

1 mnrgfsrksh tflpkiffrk msssgakdkp elqfpflqde dtvatlleck tlfilrglpg 61sgkstlarvi vdkyrdgtkm vsadaykitp gargafseey krldedlaay crrrdirilv 121lddtnherer leqlfemadq yqyqvvlvep ktawrldcaq lkeknqwqls addlkklkpg 181lekdflplyf gwfltkksse tlrkagqvfl eelgnhkafk kelrqfvpgd eprekmdlvt 241yfgkrppgvl hcttkfcdyg kapgaeeyaq qdvlkksysk aftltisalf vtpkttgary 301elseqqlqlw psdvdklspt dnlprgsrah itlgcaadve avqtgldlle ilrqekggsr 361geevgelsrg klyslgngrw mltlaknmev raiftgyygk gkpvptqgsr kggalqscti 421 i

By “CNP nucleic acid molecule” (or 2′,3′-cyclic-nucleotide3′-phosphodiesterase) is meant a polynucleotide (e.g., mRNA) encoding anCNP polypeptide. An exemplary CNP nucleic acid molecule is provided atNCBI Accession No. BC011046.

1 ctccgcgcag gcgggcggcc ccggagcgct ggtgccggca gaggcggcga cggtggcgcc 61cctcctcatc atgaggcttc tcccgaaaaa gccacacatt cctgcccaag atcttcttcc 121gcaagatgtc atcctcaggg gccaaggaca agcctgagct gcagtttccc ttccttcagg 181atgaggacac agtggccacg ctgctagagt gcaagacgct cttcatcttg cgcggcctgc 241caggaagcgg caagtccacg ctggcacggg tcatcgtgga caagtaccgt gatggcacca 301agatggtgtc ggctgacgct tacaagatca cccccggcgc tcgaggagcc ttctccgagg 361agtacaagcg gctcgatgag gacctggctg cctactgccg ccgccgggac atcagaattc 421ttgtgcttga tgacaccaac cacgaacggg aacggctgga gcagctcttt gaaatggccg 481accagtacca gtaccaggtg gtgctggtgg agcccaagac ggcgtggcgg ctggactgtg 541cccagctcaa ggagaagaac cagtggcagc tgtcggctga tgacctgaag aagctgaagc 601ctgggctgga gaaggacttc ctgccgctct acttcggctg gttcctgacc aagaagagct 661ctgagaccct ccgcaaagcc ggccaggtct tcctggaaga gctggggaac cacaaggcct 721tcaagaagga gctgcgacaa ttcgtccctg gggatgagcc cagggagaag atggacttgg 781tcacctactt tggaaagaga cccccaggcg tgctgcattg cacaaccaag ttttgtgact 841acgggaaggc tcccggggca gaggagtacg ctcaacaaga tgtgttaaag aaatcttact 901ccaaggcctt cacgctgacc atctctgccc tctttgtgac acccaagacg actggggccc 961gggtggagtt aagcgagcag caactgcagt tgtggccgag tgatgtggac aagctgtcac 1021ccactgacaa cctgccgcgg gggagccgcg cccacatcac cctcggctgt gcagctgacg 1081tagaggccgt gcagacgggc cttgacctct tagagattct gcggcaggag aaggggggca 1141gccgaggcga ggaggtgggc gagctaagcc ggggcaagct ctattccttg ggcaatgggc 1201gctggatgct gaccctggcc aagaacatgg aggtcagggc catcttcacg gggtactacg 1261ggaaaggcaa acctgtgccc acgcaaggta gccggaaggg gggcgccttg cagtcctgca 1321ccatcatatg agtgttctca ccaccactta tgcccctaga agggaagggg agagggaaac 1381gtgccctctg tttgatcctt gttttgtgac attttttttt tttttttttt tactcaaagt 1441taacctacct gtaacttttt aaaaacttgt aaaataactg accctccctt cctgtccgcc 1501ctcttcccct ctaatgctca cgctcccaac acaaggtggg cagggaggca ccattcagga 1561acctggacca aagctgacga ggctgggcca agccagggat ggggccacag ccagaacccc 1621gagccctact tccaggttct ggttagctca gccccagccc agcccagctg ctctgcccag 1681agctgggtga gtggggagac acctcagagc cccgcaaaac ccactgaccg gaggcaaaag 1741gcagtggggc tgggggtagt tttccatggt cacagagaac tagtggtggc tctgagaagg 1801ggaggacctc tgggctttga ttccatctcc ttgtcttttt tctttgtttt tagagacagg 1861gtcctgctat ttcccaagct ggagtgcagt ggtgcgatca tggctcactg cagcctcgaa 1921ctcctgggct caagcaatcc tcctgagtga tcccatttct taatcagtgt agccccaaga 1981aggctggggc tatttaccag ggtagaaaaa ggagcttacc tcccaccttt ggtcctaagt 2041ccctgccccc tccccttcac accataacta ggtaacagtt tgataactag ggaagaaagc 2101agaacagtta agcagccgcc acatccccgc tggctggggg cctcactcca ggaaggggct 2161ggactggctg tcctttccag tggcctggct ccgctgtgtg gatggggaga tcggggccag 2221aggcagaacc ctggtgagga agctccagtc ctgctctcta cccagcccat cttgcctcca 2281tggtgcctct ggaggcctct gggcctcctc taacaggggc tggtgggcac caagagccaa 2341tggagtagac ccctggctgg taagggccaa gtcccaccgg ttgcttctgg gaaggggttt 2401ctaacactag tctgtgtgct gtggttcctg gggtgccctc cactgccctc tgttcagtaa 2461cagggccttg ctaatcgggt tgtcactcaa caaaagtgct ttggatttaa gttactatcc 2521tggctttgcc caacctcagc aacctgtaag actgataatg aaataaatca tgttaatcct 2581agcaaaaaaa aaaaaaaa

By “MBP polypeptide” (or myelin basic protein) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. P02686.

1 mgnhagkrel naekastnse tnrgesekkr nlgelsrtts ednevfgead anqnngtssq 61dtavtdskrt adpknawqda hpadpgsrph lirlfsrdap gredntfkdr psesdelqti 121qedsaatses ldvmasqkrp sqrhgskyla tastmdharh gflprhrdtg ildsigrffg 181gdrgapkrgs gkdshhpart ahygslpqks hgrtqdenpv vhffknivtp rtpppsqgkg 241rglslsrfsw gaegqrpgfg yggrasdyks ahkgfkgvda qgtlskifkl ggrdsrsgsp 301marr

By “MBP nucleic acid molecule” (or myelin basic protein) is meant apolynucleotide (e.g., mRNA) encoding an MBP polypeptide. An exemplaryMBP nucleic acid molecule is provided at NCBI Accession No. M13577.

1 gaaaacagtg cagccacctc cgagagcctg gatgtgatgg cgtcacagaa gagaccctcc 61cagaggcacg gatccaagta cctggccaca gcaagtacca tggaccatgc caggcatggc 121ttcctcccaa ggcacagaga cacgggcatc cttgactcca tcgggcgctt ctttggcggt 181gacaggggtg cgccaaagcg gggctctggc aaggactcac accacccggc aagaactgct 241cactatggct ccctgcccca gaagtcacac ggccggaccc aagatgaaaa ccccgtagtc 301cacttcttca agaacattgt gacgcctcgc acaccacccc cgtcgcaggg aaaggggaga 361ggactgtccc tgagcagatt tagctggggg gccgaaggcc agagaccagg atttggctac 421ggaggcagag cgtccgacta taaatcggct cacaagggat tcaagggagt cgatgcccag 481ggcacgcttt ccaaaatttt taagctggga ggaagagata gtcgctctgg atcacccatg 541gctagacgct gaaaacccac ctggttccgg aatcctgtcc tcagcttctt aatataactg 601ccttaaaact ttaatcccac ttgcccctgt tacctaatta gagcagatga cccctcccct 661aatgcctgcg gagttgtgca cgtagtaggg tcaggccacg gcagcctacc ggcaatttcc 721ggccaacagt taaatgagaa catgaaaaca gaaaacggtt aaaactgtcc ctttctgtgt 781gaagatcacg ttccttcccc cgcaatgtgc ccccagacgc acgtgggtct tcagggggcc 841aggtgcacag acgtccctcc acgttcaccc ctccaccctt ggactttctt ttcgccgtgg 901ctcggcaccc ttgcgctttt gctggtcact gccatggagg cacacagctg cagagacaga 961gaggacgtgg gcggcagaga ggactgttga catccaagct tcctttgttt ttttttcctg 1021tccttctctc acctcctaaa gtagacttca tttttcctaa caggattaga cagtcaagga 1081gtggcttact acatgtggga gctttttggt atgtgacatg cgggctgggc agctgttaga 1141gtccaacgtg gggcagcaca gagagggggc cacctcccca ggccgtggct gcccacacac 1201cccaattagc tgaattcgcg tgtggcagag ggaggaaaag gaggcaaacg tgggctgggc 1261aatggcctca cataggaaac agggtcttcc tggagatttg gtgatggaga tgtcaagcag 1321gtggcctctg gacgtcaccg ttgccctgca tggtggcccc agagcagcct ctatgaacaa 1381cctcgtttcc aaaccacagc ccacagccgg agagtccagg aagacttgcg cactcagagc 1441agaagggtag gagtcctcta gacagcctcg cagccgcgcc agtcgcccat agacactggc 1501tgtgaccggg cgtgctggca gcggcagtgc acagtggcca gcactaaccc tccctgagaa 1561gataaccggc tcattcactt cctcccagaa gacgcgtggt agcgagtagg cacaggcgtg 1621cacctgctcc cgaattactc accgagacac acgggctgag cagacggccc ctgtgatgga 1681gacaaagagc tcttctgacc atatccttct taacacccgc tggcatctcc tttcgcgcct 1741ccctccctaa cctactgacc caccttttga ttttagcgca cctgtgattg ataggccttc 1801caaagagtcc cacgctggca tcaccctccc cgaggacgga gatgaggagt agtcagcgtg 1861atgccaaaac gcgtcttctt aatccaattc taattctgaa tgtttcgtgt gggcttaata 1921ccatgtctat taatatatag cctcgatgat gagagagtta caaagaacaa aactccagac 1981acaaacctcc aaatttttca gcagaagcac tctgcgtcgc tgagctgagg tcggctctgc 2041gatccatacg tggccgcacc cacacagcac gtgctgtgac gatggctgaa cggaaagtgt 2101acactgttcc tgaatattga aataaaacaa taaactttt

By “TUBIII polypeptide” (or TUBB3, tubulin beta chain 3) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. NP_001184110.

1 mdsvrsgafg hlfrpdnfif gqsgagnnwa kghytegael vdsvldvvrk ecencdclqg 61fqlthslggg tgsgmgtlli skvreeypdr imntfsvvps pkvsdtvvep ynatlsihql 121ventdetyci dnealydicf rtlklatpty gdlnhlvsat msgvttslrf pgqlnadlrk 181lavnmvpfpr lhffmpgfap ltargsqqyr altvpeltqq mfdaknmmaa cdprhgrylt 241vatvfrgrms mkevdeqmla iqsknssyfv ewipnnvkva vcdipprglk msstfignst 301aiqelfkris eqftamfrrk aflhwytgeg mdemefteae snmndlvsey qqyqdataee 361egemyeddee eseaqgpk

By “TUBIII nucleic acid molecule” (or TUBB3, tubulin beta chain 3) ismeant a polynucleotide (e.g., mRNA) encoding an TUBIII polypeptide. Anexemplary TUBIII nucleic acid molecule is provided at NCBI Accession No.BC000748.

1 gcccggcccg cccgcgcccg tccgcagccg cccgccagac gcgcccagta tgagggagat 61cgtgcacatc caggccggcc agtgcggcaa ccagatcggg gccaagttct gggaagtcat 121cagtgatgag catggcatcg accccagcgg caactacgtg ggcgactcgg acttgcagct 181ggagcggatc agcgtctact acaacgaggc ctcttctcac aagtacgtgc ctcgagccat 241tctggtggac ctggaacccg gaaccatgga cagtgtccgc tcaggggcct ttggacatct 301cttcaggcct gacaatttca tctttggtca gagtggggcc ggcaacaact gggccaaggg 361tcactacacg gagggggcgg agctggtgga ttcggtcctg gatgtggtgc ggaaggagtg 421tgaaaactgc gactgcctgc agggcttcca gctgacccac tcgctggggg gcggcacggg 481ctccggcatg ggcacgttgc tcatcagcaa ggtgcgtgag gagtatcccg accgcatcat 541gaacaccttc agcgtcgtgc cctcacccaa ggtgtcagac acggtggtgg agccctacaa 601cgccacgctg tccatccacc agctggtgga gaacacggat gagacctact gcatcgacaa 661cgaggcgctc tacgacatct gcttccgcac cctcaagctg gccacgccca cctacgggga 721cctcaaccac ctggtatcgg ccaccatgag cggagtcacc acctccttgc gcttcccggg 781ccagctcaac gctgacctgc gcaagctggc cgtcaacatg gtgcccttcc cgcgcctgca 841cttcttcatg cccggcttcg cccccctcac agcccggggc agccagcagt accgggccct 901gaccgtgccc gagctcaccc agcagatgtt cgatgccaag aacatgatgg ccgcctgcga 961cccgcgccac ggccgctacc tgacggtggc caccgtgttc cggggccgca tgtccatgaa 1021ggaggtggac gagcagatgc tggccatcca gagcaagaac agcagctact tcgtggagtg 1081gatccccaac aacgtgaagg tggccgtgtg tgacatcccg ccccgcggcc tcaagatgtc 1141ctccaccttc atcgggaaca gcacggccat ccaggagctg ttcaagcgca tctccgagca 1201gttcacggcc atgttccggc gcaaggcctt cctgcactgg tacacgggcg agggcatgga 1261cgagatggag ttcaccgagg ccgagagcaa catgaacgac ctggtgtccg agtaccagca 1321gtaccaggac gccacggccg aggaagaggg cgagatgtac gaagacgacg aggaggagtc 1381ggaggcccag ggccccaagt gaagctgctc gcagctggag tgagaggcag gtggcggccg 1441gggccgaagc cagcagtgtc taaacccccg gagccatctt gctgccgaca ccctgctttc 1501ccctcgccct agggctccct tgccgccctc ctgcagtatt tatggcctcg tcctccccac 1561ctaggccacg tgtgagctgc tcctgtctct gtcttattgc agctccaggc ctgacgtttt 1621acggttttgt tttttactgg tttgtgttta tattttcggg gatacttaat aaatctattg 1681ctgtcagata cccttaaaaa aaaaaaaaaa aaaaaaaaaa

By “NEUN polypeptide” (or Feminizing Locus on X-3, Fox-3, RNA-bindingprotein fox-1 homolog 3, or Hexaribonucleotide Binding Protein-3) ismeant a polypeptide or fragment thereof having at least about 85% aminoacid identity to NCBI Accession No. NP_001076044.

1 maqpyppaqy ppppqngipa eyapppphpt qdysgqtpvp tehgmtlytp aqthpeqpgs 61eastqpiagt qtvpqtdeaa qtdsqplhps dptekqqpkr lhvsnipfrf rdpdlrqmfg 121qfgkildvei ifnergskgf gfvtfetssd adrareklng tivegrkiev nnatarvmtn 181kktgnpytng wklnpvvgav ygpefyavtg fpypttgtav ayrgahlrgr gravyntfra 241apppppipty gavvyqdgfy gaeiyggyaa yryaqpaaaa aaysdsygry yaaadpyhht 301igpaatysig tm

By “NEUN nucleic acid molecule” (or Feminizing Locus on X-3, Fox-3,RNA-binding protein fox-1 homolog 3, or Hexaribonucleotide BindingProtein-3) is meant a polynucleotide (e.g., mRNA) encoding an NEUNpolypeptide. An exemplary NEUN nucleic acid molecule is provided at NCBIAccession No. NM_001082575.

1 gatacagcag cagctggtgc tcctggccag gctgtgcgtg ctctctctgc ctctctctct 61cggactctct gctctctctc tctgactctc tcctctctct ctgttggcct ggtgaaatgt 121tcttggctgt aggcacacag agccttggac tcaaggctgt tggagtcgag gacaccttga 181cttcggtcct ggaggttgaa attctgcctc tgagaagcta acagtcttcc tgtggtcgcc 241actcctcccc agcagccccc tccttgccaa ggacggtcca gaaggagccc cactggggcc 301tccccgctca gcaaagcaga cctcacctcc cactaccagc ttgaagtcac agcagccaga 361ggaaattctg ccaccatttt cccaggtctg cagcccctcc agctgggaac ctgctcctgg 421agccatccct ctgcaaacag agagcccaga gtgcctcggg gaaaattggc tgaataaaag 481agcgatcagg acgccacggc tccgcctgaa gcgatggccc agccctaccc ccccgcccag 541tacccccctc cgccacagaa cggcatccct gccgagtacg ccccgccccc accgcacccc 601acgcaggact actccggcca gaccccggtc cccacagagc atggcatgac cctgtacaca 661ccagcacaga cccaccccga gcagccaggc tccgaggcca gcacacagcc catcgccggg 721acccagacag tgccgcagac agacgaggcg gcacagacgg acagccagcc gctccacccc 781tccgacccta cagagaagca gcagcccaag cggctacacg tctccaacat ccccttccgg 841ttcagggacc ccgacttgcg gcaaatgttc gggcaattcg gaaaaatttt agacgtggag 901atcattttta acgagcgggg ctccaagggt tttgggtttg taacttttga aactagctca 961gatgctgacc gagcccggga gaagctgaat gggacgatcg tagagggacg gaaaattgag 1021gtcaataatg ccacggcccg agtgatgacc aacaagaaga cggggaaccc ctacaccaac 1081ggctggaagc taaatccagt ggtcggcgca gtctacgggc ctgaattcta tgcagtgacg 1141gggttcccct accccaccac cggcacagcc gttgcctacc ggggcgcaca tcttcggggc 1201cggggccggg ccgtgtataa tacatttcgg gctgcgccac ccccaccccc catcccgact 1261tacggagcgg tcgtgtatca ggatggattt tatggtgctg agatttatgg aggctacgca 1321gcctacagat acgctcagcc cgctgcagcg gcggcagcct acagcgacag ttacggcaga 1381gtctacgcag ctgccgaccc gtaccatcac accatcgggc ccgcggcgac ctacagcatt 1441ggaaccatgt gaaaccttcc accgtttcct tctcggacca tgaagggcaa aaacaaaaaa 1501acaaaaaaaa tcacaaaaca aaaaaaacaa aaaaagatgt taagatccaa gcaacaaaaa 1561aaaaaccaac caaaccaaga ggcatccaac caagtccaag tcccgcgtcc tggccacacg 1621cccgcaccga gggagcacgc cggcaggggc gccgaggagc ggccccagga caggacggcc 1681ccaccgcgtc ctggctggca gcacagtggg aacacgcccc tccgtctcag gcagtggggg 1741agttggaggg gaaggggcct cccttgtggg acccgtgggg ggctctgttt tccatccagt 1801cttcctttcc cagcccccaa ctcccaagac agacagtgtg gagcccagcg gcggcggagc 1861aggcccgggc ctgagcaggc aggcgctgct agcaagactt gatctttgtg gccagctgtg 1921ccagggggcc ggcggggctg aggggtgcgg gcagctttca tcccaggggc tccactgggc 1981cccgtcaccc tcctgtcgcg tcccctgcgt cccacctccc tcctgcccgg cagtcccgcc 2041cgtgccccca gcctggcgag gaagccgtcc aacagtagcc ccggggccag ctcccaacag 2101aaagggctga cgtggctcca ggactcaggg gcgctccatg ggaggacgaa ggaagcccag 2161ccagccagga gccactcctc acacctccaa gtgtggccaa gtgggccctg aggccaagga 2221cttacttgct cttcctggcc atctctccct ttctggagga ggcccggggc ctgtgtacac 2281caaggctgac ctcgtgctgc ctgctgggac ccagccctcc ctgccgctcc cctgtgagcc 2341cagtccaccg tgggcgccca gggccaggga cgggccagcg cccggctgca tcgcgaggtt 2401gggagtcaca gtggctgtgg gcctggacgg gcacagccag agcaggggcc catgggaagg 2461gcaagggatg gggaagcctg ggccggcccc ttccctgctc ccaaggcagg tgtccaggtg 2521gcgggagcag caccaaggac agccaggctt acccggtggg aggagcagga gcagagcagg 2581tggcagggag gaacccctgg cgaggcaggg agcactgaag tagggaagca gcaaaaaata 2641caggctccca acgtggctcc actgtctcat gaagtgtcaa aaatttaaaa atacacctca 2701ctttctattc agcatcagct attgaaatgg aattctcctt ttctattccc gttgtacata 2761gccccacgcc ctgcctccgg ctttgtcctc tgtacagagc cccctgtccc ctctgctgtt 2821ccggaccctt ttcttgcagc agctcaaccc cccgactcac tcagatcccc aggactgcag 2881ccgagccccg ggcttccttt cttaccattc tgtatgcttc caaggtgtga ccattcaaac 2941taacagtatt attaagatta ttaataaaga tttctttctt caaaccagga aaaaaaaaaa 3001aaaaaaa

By “SLC1A6 polypeptide” (or Excitatory amino acid transporter 4;Sodium-dependent glutamate/aspartate transporter; Solute carrier family1 member 6) is meant a polypeptide or fragment thereof having at leastabout 85% amino acid identity to NCBI Accession No. P48664.

1 msshgnslfl resgqrlgry gwlqrlqesl qqralrtrlr lqtmtlehvl rflrrnafil 61ltvsavvigv slafalrpyq ltyrqikyfs fpgellmrml qmlvlplivs slvtgmasld 121nkatgrmgmr aavyymvtti iavfigilmv tiihpgkgsk eglhregrie tiptadafmd 181lirnmfppnl veacfkqfkt qystrvvtrt mvrtengsep gasmpppfsv engtsflenv 241tralgtlqem lsfeetvpvp gsanginalg lvvfsvafgl viggmkhkgr vlrdffdsln 301eaimrlvgii iwyapvgilf liagkileme dmavlggqlg mytltvivgl flhagivlpl 361iyflvthrnp fpfiggmlqa litamgtsss satlpitfrc leeglgvdrr itrfvlpvga 421tvnmdgtaly ealaaifiaq vnnyelnlgq ittisitata asvgaagipq aglvtmvivl 481tsvglptedi tliiavdwfl drlrtmtnvl gdsigaavie hlswelelq eaeltlpslg 541kpykslmaqe kgasrgrggn esam

By “SLC1A6 nucleic acid molecule” (or Excitatory amino acid transporter4; Sodium-dependent glutamate/aspartate transporter; Solute carrierfamily 1 member 6) is meant a polynucleotide (e.g., mRNA) encoding anSLC1A6 polypeptide. An exemplary SLC1A6 nucleic acid molecule isprovided at NCBI Accession No. BC040604.

1 ggcatagcgc gtcccggctc cgcgccggtg cctccacggt ccggtccccg cgccggtgct 61gcacagtccc tggcgggtcc ccgcggcccc ggccgggcgc ttcgccgggc tccggctcct 121gcatccgggc gcagcgcgca ggccgaggcg cgggcaggcc gcccccgccg ctccggacgc 181cgggatgtaa gaggctccga aaagcagccc acgcatctca tcagatctaa gtgtctagag 241gtcgggagaa ccaagtggga aagacccacc ctcacccctc accttgtaga aactgggaac 301actagaaggg acattttctg agcaggaaac ccaagagaca gggttttacg ctgtcaccca 361agttggagtg cagtggtacg atcatagctc attgcagcct caaactcctg ggttcaagcg 421atcctcctgc tttagcctct tgagtagcta ggactacagg cacaggccac cgtgcctggc 481taatttttaa tttttaaaaa agagacaggg tctggctatg ttgcccaggc tggccatgaa 541ctcctgggct caagcggttc tccagccttc acctcccaaa gtgttgggat tgcaggcatg 601agccactgcg tctggcccac agatgctaag tgctgtctgc tcttctccag gggtcagcaa 661attttttcag caaatggccc aagagtaaat attttgagct ttgtggcccg tacaatctct 721gtcccaacaa ctcaactcag gcattgtagc ttgaaagcag ctgtagacaa taggtaatcc 781atgagtgtgg ctgtgtgcca ataaaacttt atttacaaaa acaagcagta ggctgaattt 841gactagcaga ccatagtttg tcaataccgt attatgtctt gtaaggaaga gaaaggaacc 901agacaaaact ctagcctcgg gagttttcct gactgttcag atcttagctg aatgatctcc 961cttggtatct acaggcaact tcctgctgtg gcttagggac tggaaacata atatcccaga 1021gggattccct gtgtagtctg tggttcactc tttgggattt tttttttttt tttcacagca 1081aggagaagca gcattgtggt ttcaggagat gggtccattt ggagcaggat cctaagtggg 1141gcttggcatt gggaatttgg attagctcta gaggacgcag gatctggaaa atcagggcag 1201atttcccatc ccttggatat ggtggggagt tgaggagggc aaggaagatc ccagaaaagc 1261cagtggcagc aaaacacaaa ggccagggac ctacgtactg gtaaaactga gacctccaag 1321aaacctgcag ctcgacctgg ttgaattcag atagaccatg agcagccatg gcaacagcct 1381gttccttcgg gagagcggcc agcggctggg ccgggtgggc tggctgcagc ggctgcagga 1441aagcctgcag cagagagcac tgcgcacgcg cctgcgcctg cagaccatga ccctcgagca 1501cgtgctgcgc ttcctgcgcc gaaacgcctt cattctgctg acggtcagcg ccgtggtcat 1561tggggtcagc ctggcctttg ccctgcgccc atatcagctc acctaccgcc agatcaagta 1621cttctctttt cctggagagc ttctgatgag gatgctgcag atgctggtgt tacctctcat 1681tgtctccagc ctggtcacag gtatggcatc cctggacaac aaggccacgg ggcggatggg 1741gatgcgggca gctgtgtact acatggtgac caccatcatc gcggtcttca tcggcatcct 1801catggtcacc atcatccatc ccgggaaggg ctccaaggag gggctgcacc gggagggccg 1861gatcgagacc atccccacag ctgatgcctt catggacctg atcagaaata tgtttccacc 1921aaaccttgtg gaggcctgct tcaaacagtt caagacgcag tacagcacga gggtggtaac 1981caggaccatg gtgaggacag agaacgggtc tgagccgggt gcctccatgc ctcctccatt 2041ctcagtggag aacggaacca gcttcctgga aaatgtcact cgggccttgg gtaccctgca 2101ggagatgctg agctttgagg agactgtacc cgtgcctggc tccgccaatg gcatcaacgc 2161cctgggcctc gtggtcttct ctgtggcctt tgggctggtc attggtggca tgaaacacaa 2221gggcagagtc ctcagggact tcttcgacag cctcaatgag gctattatga ggctggtggg 2281catcattatc tggtgagtcc tggtctgtgc ccacgggaag gtggagccag agctgggaag 2341tcaggctgtg gggaagctgc cgaagggctt gctggggacc tttggtcatt catttacgta 2401ttgggtgatt cacttaccca ctcaccaact cattcattca tgtctttctg ggatgatttc 2461atcactagtt cacttccttg ttcatctgtt cattcattca ttcttctatg cattggttag 2521ttcatggaat atctcactct ttcattcatt catgtccttc tgcaatgatt cattcactgc 2581tttgttcatc tgttcattca ctcattcttc tatgcattga tgaaatcact cattcagtga 2641tttattcatc tatactcatg cttcaatgca ttgatttact catttcctca tgcatttatt 2701cattcatcta tgcattggtt aaatcactgg ccaactcact aactcattca ttcattcaca 2761cttttctgca atgatttgtt cacttgttca ctcccttgct tatctgttca ttcactcatt 2821cttcaataca ttgaccaagc cattcactga catttattca gctacattta ttctttcatg 2881cattggtctg gatttatttg gtcattcatt tatttatttt gcaaaattaa tgtattttta 2941attgacaaat aaaaactgta tatattttca tgtgcaaaaa aaaaaaaaaa

By “NOGOA polypeptide” (or neurite outgrowth inhibitor A; neuriteoutgrowth inhibitor isoform A; human reticulon-4; human reticulon-4isoform A) is meant a polypeptide or fragment thereof having at leastabout 85% amino acid identity to NCBI Accession No. NP_065393.

1 medldqsplv sssdspprpq pafkyqfvre pedeeeeeee eeedededle elevlerkpa 61aglsaapvpt apaagaplmd fgndfvppap rgplpaappv aperqpswdp spvsstvpap 121splsaaaysp sklpeddepp arppppppas vspqaepvwt ppapapaapp stpaapkrrg 181ssgsvdetlf alpaasepvi rssaenmdlk eqpgntisag qedfpsvlle taaslpslsp 241lsaasfkehe ylgnlstvlp tegtlqenvs easkevseka ktllidrdlt efseleysem 301gssfsyspka esavivanpr eeiivknkde eeklvsnnil hnqqelptal tklykedevv 361ssekakdsfn ekrvaveapm reeyadfkpf ervwevkdsk edsdmlaagg kiesnleskv 421dkkcfadsle qtnhekdses snddtsfpst pegikdrsga yitcapfnpa atesiatnif 481pllgdptsen ktdelddeek kaqivteknt stktsnpflv aaqdsetdyv ttdnitkvte 541evvanmpegl tpdlvqeace selnevtgtk iayetkmdlv qtsevmqesl ypaaqlcpsf 601eeseatpspv 1pdivmeapl nsavpsagas viqpsssple assvnyesik hepenpppye 661eamsyslkkv sgikeeikep eninaalqet eapyisiacd liketklsae papdfsdyse 721makveqpvpd hselvedssp dsepvdlfsd dsipdvpqkq detvmlvkes ltetsfesmi 781eyenkeklsa lppeggkpyl esfklsldnt kdtllpdevs tlskkekipl qmeelstavy 841snddlfiske aqiretetfs dsspieiide fptlissktd sfsklareyt dlevshksei 901anapdgagsl pctelphdls lkniqpkvee kisfsddfsk ngsatskyll 1ppdvsalat 961qaeiesivkp kvlvkeaekk 1psdtekedr spsaifsael sktsvvdlly wrdikktgvv 1021fgaslfllls ltvfsivsvt ayialallsv tisfriykgv iqaiqksdeg hpfraylese 1081vaiseelvqk ysnsalghvn ctikelrrlf lvddlvdslk favlmwvfty vgalfng1t1 1141lilalislfs vpviyerhqa qidhylglan knvkdamaki qakipglkrk ae

By “NOGOA nucleic acid molecule” (or neurite outgrowth inhibitor A;neurite outgrowth inhibitor isoform A; human reticulon-4; humanreticulon-4 isoform A) is meant a polynucleotide encoding an NOGOApolypeptide. An exemplary NOGOA nucleic acid molecule (e.g., mRNA) isprovided at NCBI Accession No. NM_020532.

1 agtccctgcc ctcccctggg gagggtgagt cacgccaaac tgggcggaga gtccgctggc 61ctcactccta gctcatctgg gcggcggcgg caagtgggga cagggcgggt ggcgcatcac 121cggcgcggag gcaggaggag cagtctcatt gttccgggag ccgtcaccac agtaggtccc 181tcggctcagt cggcccagcc cctctcagtc ctccccaacc cccacaaccg cccgcggctc 241tgagacgcgg ccccggcggc ggcggcagca gctgcagcat catctccacc ctccagccat 301ggaagacctg gaccagtctc ctctggtctc gtcctcggac agcccacccc ggccgcagcc 361cgcgttcaag taccagttcg tgagggagcc cgaggacgag gaggaagaag aggaggagga 421agaggaggac gaggacgaag acctggagga gctggaggtg ctggagagga agcccgccgc 481cgggctgtcc gcggccccag tgcccaccgc ccctgccgcc ggcgcgcccc tgatggactt 541cggaaatgac ttcgtgccgc cggcgccccg gggacccctg ccggccgctc cccccgtcgc 601cccggagcgg cagccgtctt gggacccgag cccggtgtcg tcgaccgtgc ccgcgccatc 661cccgctgtct gctgccgcag tctcgccctc caagctccct gaggacgacg agcctccggc 721ccggcctccc cctcctcccc cggccagcgt gagcccccag gcagagcccg tgtggacccc 781gccagccccg gctcccgccg cgcccccctc caccccggcc gcgcccaagc gcaggggctc 841ctcgggctca gtggatgaga ccctttttgc tcttcctgct gcatctgagc ctgtgatacg 901ctcctctgca gaaaatatgg acttgaagga gcagccaggt aacactattt cggctggtca 961agaggatttc ccatctgtcc tgcttgaaac tgctgcttct cttccttctc tgtctcctct 1021ctcagccgct tctttcaaag aacatgaata ccttggtaat ttgtcaacag tattacccac 1081tgaaggaaca cttcaagaaa atgtcagtga agcttctaaa gaggtctcag agaaggcaaa 1141aactctactc atagatagag atttaacaga gttttcagaa ttagaatact cagaaatggg 1201atcatcgttc agtgtctctc caaaagcaga atctgccgta atagtagcaa atcctaggga 1261agaaataatc gtgaaaaata aagatgaaga agagaagtta gttagtaata acatccttca 1321taatcaacaa gagttaccta cagctcttac taaattggtt aaagaggatg aagttgtgtc 1381ttcagaaaaa gcaaaagaca gttttaatga aaagagagtt gcagtggaag ctcctatgag 1441ggaggaatat gcagacttca aaccatttga gcgagtatgg gaagtgaaag atagtaagga 1501agatagtgat atgttggctg ctggaggtaa aatcgagagc aacttggaaa gtaaagtgga 1561taaaaaatgt tttgcagata gccttgagca aactaatcac gaaaaagata gtgagagtag 1621taatgatgat acttctttcc ccagtacgcc agaaggtata aaggatcgtt caggagcata 1681tatcacatgt gctcccttta acccagcagc aactgagagc attgcaacaa acattatcc 1741tttgttagga gatcctactt cagaaaataa gaccgatgaa aaaaaaatag aagaaaagaa 1801ggcccaaata gtaacagaga agaatactag caccaaaaca tcaaaccctt ttcttgtagc 1861agcacaggat tctgagacag attatgtcac aacagataat ttaacaaagg tgactgagga 1921agtcgtggca aacatgcctg aaggcctgac tccagattta gtacaggaag catgtgaaag 1981tgaattgaat gaagttactg gtacaaagat tgcttatgaa acaaaaatgg acttggttca 2041aacatcagaa gttatgcaag agtcactcta tcctgcagca cagctttgcc catcatttga 2101agagtcagaa gctactcctt caccagtttt gcctgacatt gttatggaag caccattgaa 2161ttctgcagtt cctagtgctg gtgcttccgt gatacagccc agctcatcac cattagaagc 2221ttcttcagtt aattatgaaa gcataaaaca tgagcctgaa aaccccccac catatgaaga 2281ggccatgagt gtatcactaa aaaaagtatc aggaataaag gaagaaatta aagagcctga 2341aaatattaat gcagctcttc aagaaacaga agctccttat atatctattg catgtgattt 2401aattaaagaa acaaagcttt ctgctgaacc agctccggat ttctctgatt attcagaaat 2461ggcaaaagtt gaacagccag tgcctgatca ttctgagcta gttgaagatt cctcacctga 2521ttctgaacca gttgacttat ttagtgatga ttcaatacct gacgttccac aaaaacaaga 2581tgaaactgtg atgcttgtga aagaaagtct cactgagact tcatttgagt caatgataga 2641atatgaaaat aaggaaaaac tcagtgcttt gccacctgag ggaggaaagc catatttgga 2701atcttttaag ctcagtttag ataacacaaa agataccctg ttacctgatg aagtttcaac 2761attgagcaaa aaggagaaaa ttcctttgca gatggaggag ctcagtactg cagtttattc 2821aaatgatgac ttatttattt ctaaggaagc acagataaga gaaactgaaa cgttttcaga 2881ttcatctcca attgaaatta tagatgagtt ccctacattg atcagttcta aaactgattc 2941attttctaaa ttagccaggg aatatactga cctagaagta tcccacaaaa gtgaaattgc 3001taatgccccg gatggagctg ggtcattgcc ttgcacagaa ttgccccatg acctttcttt 3061gaagaacata caacccaaag ttgaagagaa aatcagtttc tcagatgact tttctaaaaa 3121tgggtctgct acatcaaagg tgctcttatt gcctccagat gtttctgctt tggccactca 3181agcagagata gagagcatag ttaaacccaa agttcttgtg aaagaagctg agaaaaaact 3241tccttccgat acagaaaaag aggacagatc accatctgct atattttcag cagagctgag 3301taaaacttca gttgttgacc tcctgtactg gagagacatt aagaagactg gagtggtgtt 3361tggtgccagc ctattcctgc tgctttcatt gacagtattc agcattgtga gcgtaacagc 3421ctacattgcc ttggccctgc tctctgtgac catcagcttt aggatataca agggtgtgat 3481ccaagctatc cagaaatcag atgaaggcca cccattcagg gcatatctgg aatctgaagt 3541tgctatatct gaggagttgg ttcagaagta cagtaattct gctcttggtc atgtgaactg 3601cacgataaag gaactcaggc gcctcttctt agttgatgat ttagttgatt ctctgaagtt 3661tgcagtgttg atgtgggtat ttacctatgt tggtgccttg tttaatggtc tgacactact 3721gattttggct ctcatttcac tcttcagtgt tcctgttatt tatgaacggc atcaggcaca 3781gatagatcat tatctaggac ttgcaaataa gaatgttaaa gatgctatgg ctaaaatcca 3841agcaaaaatc cctggattga agcgcaaagc tgaatgaaaa cgcccaaaat aattagtagg 3901agttcatctt taaaggggat attcatttga ttatacgggg gagggtcagg gaagaacgaa 3961ccttgacgtt gcagtgcagt ttcacagatc gttgttagat ctttattttt agccatgcac 4021tgttgtgagg aaaaattacc tgtcttgact gccatgtgtt catcatctta agtattgtaa 4081gctgctatgt atggatttaa accgtaatca tatctttttc ctatctatct gaggcactgg 4141tggaataaaa aacctgtata ttttactttg ttgcagatag tcttgccgca tcttggcaag 4201ttgcagagat ggtggagcta gaaaaaaaaa aaaaaaagcc cttttcagtt tgtgcactgt 4261gtatggtccg tgtagattga tgcagatttt ctgaaatgaa atgtttgttt agacgagatc 4321ataccggtaa agcaggaatg acaaagcttg cttttctggt atgttctagg tgtattgtga 4381cttttactgt tatattaatt gccaatataa gtaaatatag attatatatg tatagtgttt 4441cacaaagctt agacctttac cttccagcca ccccacagtg cttgatattt cagagtcagt 4501cattggttat acatgtgtag ttccaaagca cataagctag aagaagaaat atttctagga 4561gcactaccat ctgttttcaa catgaaatgc cacacacata gaactccaac atcaatttca 4621ttgcacagac tgactgtagt taattttgtc acagaatcta tggactgaat ctaatgcttc 4681caaaaatgtt gtttgtttgc aaatatcaaa cattgttatg caagaaatta ttaattacaa 4741aatgaagatt tataccattg tggtttaagc tgtactgaac taaatctgtg gaatgcattg 4801tgaactgtaa aagcaaagta tcaataaagc ttatagactt aaaaaaaaaa aaaaaaaaaa 4861aaaaaaaaaa a

By “oligodendrocyte 01 polypeptide” (or oligodendrocyte marker 01;oligodendrocyte transcription factor 1; olig1) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. Q8TAK6.

1 myyavsqarv navpgtmlrp qrpgdlqlga slyelvgyrq ppssssssts stsstsssst 61tapllpkaar ekpeapaepp gpgpgsgahp ggsarpdake eqqqqlrrki nsrerkrmqd 121lnlamdalre vilpysaahc qgapgrklsk iatlllarny illlgsslqe lrralgegag 181paaprlllag 1pllaaapgs vllapgavgp pdalrpakyl slaldeppcg qfalpgggag 241gpglctcavc kfphlvpasl glaavqaqfs k

By “oligodendrocyte 01 nucleic acid molecule” (or oligodendrocyte marker01; oligodendrocyte transcription factor 1; olig1) is meant apolynucleotide encoding an oligodendrocyte 01 polypeptide. An exemplaryoligodendrocyte 01 nucleic acid molecule (e.g., mRNA) is provided atNCBI Accession No. NM_138983.

1 gttctagatc gtttccccgc gcgcaggtcc gcggggaggg gcggcctgcc gaccggccca 61ccccagggcg ttcctgaagg gcgtcctcgg ccgcccccac cgcctcccag atgtactatg 121cggtttccca ggcgcgcgtg aacgcggtcc ccgggaccat gctgcggcca cagcggcccg 181gagacttgca gctcggggcc tccctctacg agctggtggg ctacaggcag ccgccctcct 241cctcctcctc ctccacctcc tccacctcct ccacttcctc ctcctccacg acggcccccc 301tcctccccaa ggctgcgcgc gagaagccgg aggcgccggc cgagcctcca ggccccgggc 361ccgggtcagg cgcgcacccg ggcggcagcg cccggccgga cgccaaggag gagcagcagc 421agcagctgcg gcgcaagatc aacagccgcg agcggaagcg catgcaggac ctgaacctgg 481ccatggacgc cctgcgcgag gtcatcctgc cctactcagc ggcgcactgc cagggcgcgc 541ccggccgcaa gctctccaag atagccacgc tgctgctcgc ccgcaactac atcctactgc 601tgggcagctc gctgcaggag ctgcgccgcg cgctgggcga gggcgccggg cccgccgcgc 661cgcgcctgct gctggccggg ctgcccctgc tcgccgccgc gcccggctcc gtgctgctgg 721cgcccggcgc cgtaggaccc cccgacgcgc tgcgccccgc caagtacctg tcgctggcgc 781tggacgagcc gccgtgcggc cagttcgctc tccccggcgg cggcgcaggc ggccccggcc 841tctgcacctg cgccgtgtgc aagttcccgc acctggtccc ggccagcctg ggcctggccg 901ccgtgcaggc gcaattctcc aagtgagggc gggtctgggc ctggggcgcg acctcggccc 961ggcctccctt cgctcagctt ctccgcgccc ctgctccctg cgtctgggag agcgaggccg 1021agcaaggaaa gcatttcgaa ccttccagtc cagaggaagg gactgtcggg cacccccttc 1081cccgccccca cccctgggac gttaaagtga ccagagcgga tgttcgatgg cgcctcgggg 1141cagtttgggg ttctgggtcg gttccagcgg ctttaggcag aaagtgctcg ctctcaccca 1201gcacatctct ctccttgtcc ctggagttgc gcgcttcgcg gggccgatgt agaacttagg 1261gcgccttgcc gtggttggcg cgccccgggt gcagcgagag gccatccccg agcgctacct 1321ccccggagcg gagcacgcgg gctcccagta ctaggggctg cgctcgagca gtggcggggg 1381cggaggggtg gttcttttcc ttctcctccg ccagaggcca cgggcgccct tgttcccgcc 1441ggccaggtcc tatcaaagga ggctgccgga actcaagagg cagaaaaaga ccagttaggc 1501ggtgcagacg gtctgggacg tggcagacgg acggaccctc ggcggacagg tggtcggcgt 1561cggggtgcgg tgggtagggg cgaggacaac gcagggtgcg ctgggttggg acgtgggtcc 1621acttttgtag accagctgtt tggagagctg tatttaagac tcgcgtatcc agtgttttgt 1681cgcagagagt tttcactctt aaatcctggg ggtttcttag aaagcaactt agaactcgag 1741attcaccttt cgtttccctt tccccaaaag tagcgtaacc aacatttaag cttgcttaaa 1801aacgaaaacc aaccgccttg catccagtgt tcccgattta ctaaaatagg taaccaggcg 1861tctcacagtc gccgtcctgt caagagcgct aatgaacgtt ctcattaaca cgcaggagta 1921ccgggagccc tgaaccgccc gctgctcggc ggatcccagc tgcggtggcg acggcgggaa 1981ggcgctttcc gctgttcctc agcgggccgg gcccttgacc agcgcggccc gcaggtcttc 2041cttctcgccg tcttgcagtt gaagagctac atacgtagtc agtttcgatt tgttacagac 2101gttaacaaat tcctttaccc aaggttatgc tatgaccttt ccgcagttta ctttgatttt 2161ctatgtttaa ggttttggtt gttggtagta gccgaattta actggcactt tattttactt 2221ctaaccttgt ttcctgacgg tgtacagaat caacaaaata aaacatttaa agtctgattt 2281tttaaaaaaa aaaaaaaa

By “oligodendrocyte 02 polypeptide” (or oligodendrocyte marker 02;oligodendrocyte transcription factor 2; olig2) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. Q13516.

1 mdsdaslvss rpsspepddl flparskgss gsaftggtvs sstpsdcppe lsaelrgamg 61sagahpgdkl ggsgfkssss stssstssaa asstkkdkkq mtepelqqlr lkinsrerkr 121mhdlniamdg lrevmpyahg psvrklskia tillarnyil mltnsleemk rlvseiyggh 181hagfhpsacg glahsaplpa atahpaaaah aahhpavhhp ilppaaaaaa aaaaaaayss 241aslpgsglps vgsirpphgl lkspsaaaaa plggggggsg asggfqhwgg mpcpcsmcqv 301ppphhhvsam gagslprlts dak

By “oligodendrocyte 02 nucleic acid molecule” (or oligodendrocyte marker02; oligodendrocyte transcription factor 2; olig2) is meant apolynucleotide encoding an oligodendrocyte 02 polypeptide. An exemplaryoligodendrocyte 02 nucleic acid molecule (e.g., mRNA) is provided atNCBI Accession No. NM_005806.

1 gggtgcttat tatagatcga cgcgacacca gcgcccggtg ccaggttctc ccctgaggct 61tttcggagcg agctcctcaa atcgcatcca gagtaagtgt ccccgcccca cagcagccgc 121agcctagatc ccagggacag actctcctca actcggctgt gacccagaat gctccgatac 181agggggtctg gatccctact ctgcgggcca tttctccaga gcgactttgc tcttctgtcc 241tccccacact caccgctgca tctccctcac caaaagcgag aagtcggagc gacaacagct 301ctttctgccc aagccccagt cagctggtga gctccccgtg gtctccagat gcagcacatg 361gactctgggc cccgcgccgg ctctgggtgc atgtgcgtgt gcgtgtgttt gctgcgtggt 421gtcgatggag ataaggtgga tccgtttgag gaaccaaatc attagttctc tatttagatc 481tccattctcc ccaaagaaag gccctcactt cccactcgtt tattccagcc cgggggctca 541gttttcccac acctaactga aagcccgaag cctctagaat gccacccgca ccccgagggt 601caccaacgct ccctgaaata acctgttgca tgagagcaga ggggagatag agagagctta 661attataggta cccgcgtgca gctaaaagga gggccagaga tagtagcgag ggggacgagg 721agccacgggc cacctgtgcc gggaccccgc gctgtggtac tgcggtgcag gcgggagcag 781cttttctgtc tctcactgac tcactctctc tctctctccc tctctctctc tctcattctc 841tctcttttct cctcctctcc tggaagtttt cgggtccgag ggaaggagga ccctgcgaaa 901gctgcgacga ctatcttccc ctggggccat ggactcggac gccagcctgg tgtccagccg 961cccgtcgtcg ccagagcccg atgacctttt tctgccggcc cggagtaagg gcagcagcgg 1021cagcgccttc actgggggca ccgtgtcctc gtccaccccg agtgactgcc cgccggagct 1081gagcgccgag ctgcgcggcg ctatgggctc tgcgggcgcg catcctgtgg acaagctagg 1141aggcagtggc ttcaagtcat cctcgtccag cacctcgtcg tctacgtcgt cggcggctgc 1201gtcgtccacc aagaaggaca agaagcaaat gacagagccg gagctgcagc agctgcgtct 1261caagatcaac agccgcgagc gcaagcgcat gcacgacctc aacatcgcca tggatggcct 1321ccgcgaggtc atgccgtacg cacacggccc ttcggtgcgc aagctttcca agatcgccac 1381gctgctgctg gcgcgcaact acatcctcat gctcaccaac tcgctggagg agatgaagcg 1441actggtgagc gagatctacg ggggccacca cgctggcttc cacccgtcgg cctgcggcgg 1501cctggcgcac tccgcgcccc tgcccgccgc caccgcgcac ccggcagcag cagcgcacgc 1561cgcacatcac cccgcggtgc accaccccat cctgccgccc gccgccgcag cggctgctgc 1621cgccgctgca gccgcggctg tgtccagcgc ctctctgccc ggatccgggc tgccgtcggt 1681cggctccatc cgtccaccgc acggcctact caagtctccg tctgctgccg cggccgcccc 1741gctggggggc gggggcggcg gcagtggggc gagcgggggc ttccagcact ggggcggcat 1801gccctgcccc tgcagcatgt gccaggtgcc gccgccgcac caccacgtgt cggctatggg 1861cgccggcagc ctgccgcgcc tcacctccga cgccaagtga gcctactggc gccggcgcgt 1921tctggcgaca ggggagccag gggccgcggg gaagcgagga ctggcctgcg ctgggctcgg 1981gagctctgtc gcgaggaggg gcgcaggacc atggactggg ggtggggcat ggtggggatt 2041tcagcatctg cgaacccaag caatgggggc gcccacagag cagtggggag tgaggggatg 2101ttctctccgg gacctgatcg agcgctgtct ggctttaacc tgagctggtc cagtagacat 2161cgttttatga aaaggtaccg ctgtgtgcat tcctcactag aactcatccg acccccgacc 2221cccacctccg ggaaaagatt ctaaaaactt ctttccctga gagcgtggcc tgacttgcag 2281actcggcttg ggcagcactt cgggggggga gggggtgtta tgggaggggg acacattggg 2341gccttgctcg tcttcctcct ttcttggcgg gtgggagact ccgggtagcc gcactgcaga 2401agcaacagcc cgaccgcgcc ctccagggtc gtccctggcc caaggccagg ggccacaagt 2461tagttggaag ccggcgttcg gtatcagaag cgctgatggt catatccaat ctcaatatct 2521gggtcaatcc acaccctctt agaactgtgg ccgttcctcc ctgtctctcg ttgatttggg 2581agaatatggt tttctaataa atctgtggat gttccttctt caacagtatg agcaagttta 2641tagacattca gagtagaacc acttgtggat tggaataacc caaaactgcc gatttcaggg 2701gcgggtgcat tgtagttatt attttaaaat agaaactacc ccaccgactc atctttcctt 2761ctctaagcac aaagtgattt ggttattttg gtacctgaga acgtaacaga attaaaaggc 2821agttgctgtg gaaacagttt gggttatttg ggggttctgt tggcttttta aaattttctt 2881ttttggatgt gtaaatttat caatgatgag gtaagtgcgc aatgctaagc tgtttgctca 2941cgtgactgcc agccccatcg gagtctaagc cggctttcct ctattttggt ttatttttgc 3001cacgtttaac acaaatggta aactcctcca cgtgcttcct gcgttccgtg caagccgcct 3061cggcgctgcc tgcgttgcaa actgggcttt gtagcgtctg ccgtgtaaca cccttcctct 3121gatcgcaccg cccctcgcag agagtgtatc atctgtttta tttttgtaaa aacaaagtgc 3181taaataatat ttattacttg tttggttgca aaaacggaat aaatgactga gtgttgagat 3241tttaaataaa atttaaagca aaaaaaaaaa aaaaa

By “oligodendrocyte 04 polypeptide” (or oligodendrocyte marker 04;oligodendrocyte transcription factor 4; olig4) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. Q05586.

By “oligodendrocyte 04 nucleic acid molecule” (or oligodendrocyte marker04; oligodendrocyte transcription factor 4; olig4) is meant apolynucleotide encoding an oligodendrocyte 04 polypeptide. An exemplaryoligodendrocyte 04 nucleic acid molecule (e.g., mRNA) is provided atNCBI Accession No. NM_007327.

By “GFAP” (or Glial fibrillary acidic protein) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. P14136.

1 merrritsaa rrsyvssgem mvgglapgrr lgpgtrlsla rmppplptry dfslagalna 61gfketraser aemmelndrf asyiekvrfl eqqnkalaae lnqlrakept kladvyqael 121relrlrldql tansarleve rdnlaqdlat vrqklqdetn lrleaennla ayrqeadeat 181larldlerki esleeeirfl rkiheeevre lqeqlarqqv hveldvakpd ltaalkeirt 241qyeamassnm heaeewyrsk fadltdaaar naellrqakh eandyrrqlq sltcdleslr 301gtneslerqm reqeerhvre aasyqealar leeegqslkd emarhlqeyq dllnvklald 361ieiatyrkll egeenritip vqtfsnlqir etsldtksys eghlkrnivv ktvemrdgev 421ikeskqehkd vm

By “GFAP nucleic acid molecule” (or Glial fibrillary acidic protein) ismeant a polynucleotide encoding an GFAP polypeptide. An exemplary GFAPnucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No.NM_002055.

1 gcaggatgga gaggagacgc atcacctccg ctgctcgccg ctcctacgtc tcctcagggg 61agatgatggt ggggggcctg gctcctggcc gccgtctggg tcctggcacc cgcctctccc 121tggctcgaat gccccctcca ctcccgaccc gagtggattt ctccctggct ggggcactca 181atgctggctt caaggagacc cgggccagtg agcgggcaga gatgatggag ctcaatgacc 241gctttgccag ctacatcgag aaggttcgct tcctggaaca gcaaaacaag gcgctggctg 301ctgagctgaa ccagctgcgg gccaaggagc ccaccaagct ggcagacgtc taccaggctg 361agctgcgaga gctgcggctg cggctcgatc aactcaccgc caacagcgcc cggctggagg 421ttgagaggga caatctggca caggacctgg ccactgtgag gcagaagctc caggatggaa 481ccaacctgag gctggaagcc gagaacaacc tggctgccta tagacaggaa gcagatgaag 541ccaccctggc ccgtctggat ctggagagga agattgagtc gctggaggag gagatccggt 601tcttgaggaa gatccacgag gaggaggttc gggaactcca ggagcagctg gcccgacagc 661aggtccatgt ggagcttgac gtggccaagc cagacctcac cgcagccctg aaagagatcc 721gcacgcagta tgaggcaatg gcgtccagca acatgcatga agccgaagag tggtaccgct 781ccaagtttgc agacctgaca gacgctgctg cccgcaacgc ggagctgctc cgccaggcca 841agcacgaagc caacgactac cggcgccagt tgcagtcctt gacctgcgac ctggagtctc 901tgcgcggcac gaacgagtcc ctggagaggc agatgcgcga gcaggaggag cggcacgtgc 961gggaggcggc cagttatcag gaggcgctgg cgcggctgga ggaagagggg cagagcctca 1021aggacgagat ggcccgccac ttgcaggagt accaggacct gctcaatgtc aagctggccc 1081tggacatcga gatcgccacc tacaggaagc tgctagaggg cgaggagaac cggatcacca 1141ttcccgtgca gaccttctcc aacctgcaga ttcgagaaac cagcctggac accaagtctg 1201tgtcagaagg ccacctcaag aggaacatcg tggtgaagac cgtggagatg cgggatggag 1261aggtcattaa ggagtccaag caggagcaca aggatgtgat gtgaggcagg acccacctgg 1321tggcctctgc cccgtctcat gaggggcccg agcagaagca ggatagttgc tccgcctctg 1381ctggcacatt tccccagacc tgagctcccc accaccccag ctgctcccct ccctcctctg 1441tccctaggtc agcttgctgc cctaggctcc gtcagtatca ggcctgcc

By “s100b” (or S-100 protein beta chain; S-100 protein subunit beta;S100 calcium-binding protein B) is meant a polypeptide or fragmentthereof having at least about 85% amino acid identity to NCBI AccessionNo. P04271.

1 mselekamva lidvfhqysg regdkhklkk selkelinne lshfleeike qevvdkvmet 61ldndgdgecd fqefmafvam vttacheffe he

By “s100b nucleic acid molecule” (or S-100 protein beta chain; S-100protein subunit beta; S100 calcium-binding protein B) is meant apolynucleotide encoding an s100b polypeptide. An exemplary s100b nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006272.

1 gggcagaggg aataagaggc tgcctctgcc caccagtcct gccgcccagg acccgcagca 61gagacgacgc ctgcagcaag gagaccagga aggggtgaga caaggaagag gatgtctgag 121ctggagaagg ccatggtggc cctcatcgac gttttccacc aatattctgg aagggaggga 181gacaagcaca agctgaagaa atccgaactg aaggagctca tcaacaatga gctttcccat 241ttcttagagg aaatcaaaga gcaggaggtt gtggacaaag tcatggaaac actggacaat 301gatggagacg gcgaatgtga cttccaggaa ttcatggcct ttgttgccat ggttactact 361gcctgccacg agttctttga acatgagtga gattagaaag cagccaaacc tttcctgtaa 421cagagacggt catgcaagaa agcagacagc aagggcttgc agcctagtag gagctgagct 481ttccagccgt gttgtagcta attaggaagc ttgatttgct ttgtgattga aaaattgaaa 541acctctttcc aaaggctgtt ttaacggcct gcatcattct ttctgctata ttaggcctgt 601gtgtaagctg actggcccca gggactcttg ttaacagtaa cttaggagtc aggtctcagt 661gataaagcgt gcaccgtgca gcccgccatg gccgtgtaga ccctaacccg gagggaaccc 721tgactacaga aattaccccg gggcaccctt aaaacttcca ctacctttaa aaaacaaagc 781cttatccagc attatttgaa aacactgctg ttctttaaat gcgttcctca tccatgcaga 841taacagctgg ttggccggtg tggccctgca agggcgtggt ggcttcggcc tgcttcccgg 901gatgcgcctg atcaccaggt gaacgctcag cgctggcagc gctcctggaa aaagcaactc 961catcagaact cgcaatccga gccagctctg ggggctccag cgtggcctcc gtgacccatg 1021cgattcaagt cgcggctgca ggatccttgc ctccaacgtg cctccagcac atgcggcttc 1081cgagggcact accgggggct ctgagccacc gcgagggcct gcgttcaata aaaag

By “SOX10 polypeptide” (or SRY-related HMG-box transcription factor) ismeant a polypeptide or fragment thereof having at least about 85% aminoacid identity to NCBI Accession No. NP_008872.1.

MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQDGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKYQPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPGEGSPMSDGNPEHPSGQSHGPPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNVDIGEISHEVMSNMETFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPGVDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSGPYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSH SPTHWEQPVYTTLSRP

By “SOX10 nucleic acid molecule” (or SRY-related HMG-box transcriptionfactor) is meant a polynucleotide encoding an SOX10 polypeptide. Anexemplary SOX10 nucleic acid molecule (e.g., mRNA) is provided at NCBIAccession No. NM_006941.3.

1 gtccggccag ggtggttggt ggtaaggatt caggctccgt cctaacgagg ccgtggcctg 61aggctcaggg ccccccgccc ctccctccca gcccaccagc gtcacctccc agccccgagc 121tggaccgcac accttgggac acggttttcc acttcctaag gacgagcccc agactggagg 181agaggtccga ggaggtgggc gttggactct ttgcgaggac cccggcggct ggcccggggg 241aggcggccga ggcggcggcg gcggcggccg ggggcgacat ggcggaggag caggacctat 301cggaggtgga gctgagcccc gtgggctcgg aggagccccg ctgcctgtcc ccggggagcg 361cgccctcgct agggcccgac ggcggcggcg gcggatcggg cctgcgagcc agcccggggc 421caggcgagct gggcaaggtc aagaaggagc agcaggacgg cgaggcggac gatgacaagt 481tccccgtgtg catccgcgag gccgtcagcc aggtgctcag cggctacgac tggacgctgg 541tgcccatgcc cgtgcgcgtc aacggcgcca gcaaaagcaa gccgcacgtc aagcggccca 601tgaacgcctt catggtgtgg gctcaggcag cgcgcaggaa gctcgcggac cagtacccgc 661acctgcacaa cgctgagctc agcaagacgc tgggcaagct ctggaggctg ctgaacgaaa 721gtgacaagcg ccccttcatc gaggaggctg agcggctccg tatgcagcac aagaaagacc 781acccggacta caagtaccag cccaggcggc ggaagaacgg gaaggccgcc cagggcgagg 841cggagtgccc cggtggggag gccgagcaag gtgggaccgc cgccatccag gcccactaca 901agagcgccca cttggaccac cggcacccag gagagggctc ccccatgtca gatgggaacc 961ccgagcaccc ctcaggccag agccatggcc cacccacccc tccaaccacc ccgaagacag 1021agctgcagtc gggcaaggca gacccgaagc gggacgggcg ctccatgggg gagggcggga 1081agcctcacat cgacttcggc aacgtggaca ttggtgagat cagccacgag gtaatgtcca 1141acatggagac ctttgatgtg gctgagttgg accagtacct gccgcccaat gggcacccag 1201gccatgtgag cagctactca gcagccggct atgggctggg cagtgccctg gccgtggcca 1261gtggacactc cgcctggatc tccaagccac caggcgtggc tctgcccacg gtctcaccac 1321ctggtgtgga tgccaaagcc caggtgaaga cagagaccgc ggggccccag gggcccccac 1381actacaccga ccagccatcc acctcacaga tcgcctacac ctccctcagc ctgccccact 1441atggctcagc cttcccctcc atctcccgcc cccagtttga ctactctgac catcagccct 1501caggacccta ttatggccac tcgggccagg cctctggcct ctactcggcc ttctcctata 1561tggggccctc gcagcggccc ctctacacgg ccatctctga ccccagcccc tcagggcccc 1621agtcccacag ccccacacac tgggagcagc cagtatatac gacactgtcc cggccctaaa 1681gggggccctg tcgccaccac cccccgccca gcccctgccc ccagcctgtg tgccctgttc 1741cttgcccacc tcaggcctgg tggtggcagt ggaggaggct gaggaggctg aagaggctga 1801caggtcgggg ggctttctgt ctggctcact gccctgatga cccacccgcc ccatccaggc 1861tccagcagca aagccccagg agaacaggct ggacagagga gaaggaggtt gactgttgca 1921cccacactga aagatgaggg gctgcacctt cccccaggaa tgaccctcta tcccaggacc 1981tgagaagggc ctgctcaccc tcctcgggga ggggaagcac cagggttggt ggcatcggag 2041gccttaccac tcctatgact cctgttttct ctctcacaga tagtgagggt ctgacatgcc 2101catgccacct atgccacagt gcctaagggc taggccaccc agagactgtg cccggagctg 2161gccgtgtctc ccactcaggg gctgagagta gctttgagga gcctcattgg ggagtggggg 2221gttcgaggga cttagtggag ttctcatccc ttcaatgccc cctccctttc tgaaggcagg 2281aaggagttgg cacagaggcc ccctgatcca attctgtgcc aataacctca ttctttgtct 2341gagaaacagc ccccagtcct cctccactac aacctccatg accttgagac gcatcccagg 2401aggtgacgag gcaggggctc caggaaagga atcagagaca attcacagag cctccctccc 2461tgggctcctt gccagctccc tcttccctta ctaggctcta tggcccctgc tcagtcagcc 2521ccactccctg ggcttcccag agagtgacag ctgctcaggc cctaaccctt ggctccagga 2581gacacagggc ccagcaccca ggttgctgtc ggcaggctga agacactaga atcctgacct 2641gtacattctg cccttgcctc ttaccccttg cctcccagtg gtatttgaat aaagtatgta 2701gctatatctg cccctatttt cctgttctgc agccccccaa atccacatgt aactcattac 2761tgtctcctgt tatttatctc agtagtcccc tctcctagcc actctagccc ctattaactc 2821tgcattaagc attccacata ataaaattaa aggttccggt taaaaaaaaa aaaaaaaaaa 2881aa

By “SYN1 protein” (or Synaptin I protein) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity toGenBank: AH006533.2.

MNYLRRRLSDSNFMANLPNGYMTDLQRPQPPPPPPGAHSPGATPGPGTATAERSSGVAPAASPAAPSPGSSGGGGFFSSLSNAVKQTTAAAAATFSEQVGGGSGGAGRGGAASRVLLVIDEPHTDWAKYFKGKKIHGGIDIKVEQAEFSDLNLVAHANGGFSVDMEVLRNGVKVVRSLKPDFVLIRQHAFSMARNGDYRSLVIGLQYAGIPSVNSLHSVYNFCDKPWVFAQMVRLHKKLGTEEFPLIDQTFYPNHKEMLSSTTYPVVVKMGHAHSGMGKVKVDNQHDFQDIASVVALTKTYATAEPFIDAKYDVRVQKIGQNYKAYMRTSVSGNWKTNTGSAMLEQIAMSDRYKLWVDTCSEIFGGLDICAVEALHGKDGRDHIIEVVGSSMPLIGDHQDEDKQLIVELVVNKMAQALPRQRQRDASPGRGSHGQTPSPGALPLGRQTSQQPAGPPAQQRPPPQGGPPQPGPGPQRQGPPLQQRPPPQGQQHLSGLGPPAGSPLPQRLPSPTSAPQQPASQAAPPTQGQGRQSRPVAGGPGAPPAARPPASPSPQRQAGPPQATRQTSVSGPAPPKASGAPPGGQQRQGPPQKPPGPAGPTRQASQAGPVPRTGPPTTQQPRPSGPGPAGAPKPQLAQKPSQDVPPPATAAAGGPPHPQLNKSQSLTNAFNLPEPAPPRPSLSQDEVKAETIRSLRKSFA SLFSD

By “SYN1 nucleic acid molecule” (or synapsin I gene) is meant apolynucleotide encoding an SYN1 polypeptide. An exemplary SYN1 nucleicacid molecule (e.g., mRNA) is provided at GenBank: AH006533.2.

1 ctcgagagag aaggagagga cattcctggc agaagttaca acacatgcaa aggtacagag 61gttgccccct tcctacccct ctccttagag gtgggttaga gatgtatcct ttttacagat 121gaggaaacca aatctcagaa agattaagtc actttcccaa gtgtatggtg gaggccccac 181ttgaacccag gcactgtgtc tccagacccc acactattac tgccttgttt aaaccagcca 241actgatttaa tgaataaagg atgaacaaat gaataagtgg atgagtcacc tgaaaattct 301gcaggcaaag agactccata tctacttact tcttgcctat cttctgccac ctctcctagt 361ccaccatcac tgctcactat ggtcaaggtc ctacccaatc tggcccctgc taccacaacc 421cccttcagct tgttccagcc acattggcac tggatgtttc ctcttcctgg cacattctta 481aaaaaatgtg ttgatcataa agtgaacatg accctttggg aattaactgg agttcttgta 541ttccctcatc tgtaaaatag acattatatt atccacccca ctggattgtt gtgagggtgg 601gatgaaatga tgcatgtaaa cacgcttagc ttaagagttg ggtacaatca gtgaacaaat 661gattatgaat tagtgctttt attgtagtca gaatcataaa gatttgacag gttcccatat 721cccacctctg cttggactac ctcatttgct catatgcaaa gattatttgg tacctactgt 781gtgtgcacca tgggatgggc ctgcctctgt ggaaagttct tgggtgcagg gggagacagc 841catgggcact gatgacatca ggtagttatc gtgagttttg gcggtgtcca gagcaaaggg 901atggtggcgt atataccaag tgtgttctgg tgtgggggtg gacacgcacc agggctaggg 961ctgcagagaa tgtctgtgtt gcagatctag gtttctccat gatcatcggt gggaatgtgt 1021tttgtctgca agtgtatgct catatgagtt tccctgggtc tctgtgtgtc agtgtgttac 1081ctgtgtgtgt gggggtatgg gtgtatgcat gcatgtatgt aacatgccca tgtgtgttac 1141tctggacttg tatgtctgta tgtataccta gattggcgtg tgttctgtct gtacatgccc 1201tcgtatgttt cctcactttt gtgtgtgttt atatgtgtgt catttcttgt gtgccctcca 1261ggcccccctt gccaccttgg gcaagggtgt gtacaccacc caagtgtcca cctccgcttg 1321tctgatgctg tctgtgacgc ccccgctctc tgcctagctg agcctgtgtg gatgtgggag 1381actaatctcc ccgcgggcac tgcgtgtgac ctcacccccc tctgtgaggg ggttatttct 1441ctactttcgt gtctctgagt gtgcttccag tgcccccctc cccccaaaaa atgccttctg 1501agttgaatat caacactaca aaccgagtat ctgcagactg cagagggccc tgcgtatgag 1561tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc 1621ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga 1681gggggagggg aaacaggatg cggcgaggcg cgtcgcgact gccagcttca gcaccgcgga 1741cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc 1801tgacgtcact cgccggtccc ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg 1861ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg 1921accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag 1981gagtcgtgtc gtgcctgaga gcgcagctgt gctcctgggc accgcgcagt ccgcccccgc 2041ggctcctggc cagaccaccc ctaggacccc ctgccccaag tcgcagccat gaactacctg 2101cggcgccgcc tgtcggacag caactttatg gccaatctgc caaatgggta catgacagac 2161ctgcagcgtc cgcagccgcc cccaccgccg cccggtgccc acagccccgg agccacgccc 2221ggtcccggga ccgccactgc cgagaggtcc tccggggtcg ccccagcggc ctctccggcc 2281gcccctagcc ccgggtcctc ggggggcggt ggcttcttct cgtcgctgtc caacgcggtc 2341aagcagacca cggcggcggc agctgccacc ttcagcgagc aggtgggcgg cggctctggg 2401ggcgcaggcc gcgggggagc cgcctccagg gtgctgctgg tcatcgacga gccgcacacc 2461gactggtaag

By “SYP protein” (or synaptophysin protein) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIReference Sequence: NM_003179.2.

MLLLADMDVVNQLVAGGQFRVVKEPLGFVKVLQWVFAIFAFATCGSYSGELQLSVDCANKTESDLSIEVEFEYPFRLHQVYFDAPTCRGGTTKVFLVGDYSSSAEFFVTVAVFAFLYSMGALATYIFLQNKYRENNKGPMLDFLATAVFAFMWLVSSSAWAKGLSDVKMATDPENIIKEMPVCRQTGNTCKELRDPVTSGLNTSVVFGFLNLVLWVGNLWFVFKETGWAAPFLRAPPGAPEKQPAPGDAYGDAGYGQGPGGYGPQDSYGPQGGYQPDYGQPAGSGGSGYGPQGDYGQQGY GPQGAPTSFSNQM

By “SYP nucleic acid molecule” (or synaptophysin gene) is meant apolynucleotide encoding an SYN1 polypeptide. An exemplary SYP nucleicacid molecule (e.g., mRNA) is provided at NCBI Reference Sequence:NM_003179.2.

1 gccccctgca ttgctgatgc tgctgctggc ggacatggac gtggtgaatc agctggtggc 61tgggggtcag ttccgggtgg tcaaggagcc cctcggcttt gtgaaggtgc tgcaatgggt 121cttcgccatc ttcgcctttg ccacatgcgg cagctacagt ggggagctcc agctgagcgt 181ggattgtgcc aacaagaccg agagtgacct cagcatcgag gtcgagttcg agtacccctt 241caggctgcac caagtgtact ttgatgcacc cacctgccga gggggcacca ccaaggtctt 301cttagttggg gactactcct cgtcagccga attctttgtc accgtggccg tgtttgcctt 361cctctactcc atgggggctc tggccaccta catcttcctg cagaacaagt accgagagaa 421taacaaaggg cccatgctgg actttctggc cacggctgtg ttcgccttca tgtggctagt 481tagctcatcg gcatgggcca aggggctgtc agatgtgaag atggccacag acccagagaa 541cattatcaag gagatgcctg tctgccgcca gacagggaac acatgcaagg agctgagaga 601ccctgtgacc tcgggactca acacctcggt ggtgttcggc ttcctgaacc tggtgctctg 661ggtcggcaac ctgtggttcg tgtttaagga gacaggctgg gccgccccgt tcctgcgcgc 721gcctcccggc gcccccgaga aacaaccggc acccggggac gcctacggcg atgcaggcta 781cgggcagggc cccggcgggt acgggcccca ggattcctac gggcctcagg gcggctacca 841gcctgactat ggtcaaccag ccggcagcgg tggcagtggc tacgggcctc agggcgacta 901tgggcagcaa ggctacggcc cgcagggtgc acccacctcc ttctccaatc agatgtagtc 961tggtcagtga agcccaggag gacctggggg gggcaagagc tcaggagaag gcctgccccc 1021cttcccaccc ctatacccta ggtctccacc cctcaagcca ggagaccctg tctttgctgt 1081ttatatatat atatattata tataaatatc tatttatctg tctgagccct gccctcactc 1141cactcccctc atccactagg tgcccagtct tgagtgggcc ccctctctta ccccgtccct 1201ttccctgcat cccttggccc ctctctgttt accctccctg tcccctgagg ttaaggggat 1261ctaaaaggag gacagggagg gaacagacct cggctgtgtg gggagggtgg gcgtgacttc 1321agactctctc ctctctctcc ctccactcct cccaactctg gccttggttc ctccagcaat 1381gcctgcctga acaaaggccg ttagggaaat ccaactccag ggttaaagaa aggcagagat 1441tgggggggct tggggtagag aggacagttt aggacccaag gtggtcttgg agaggaggtg 1501tggagtggag gggtcagcag gggggttggg ttccagacag agtggatctg gagtctgaag 1561gagaggagtg cgctagagca ttctggggtg gggcttggaa gggcgctgag ggcagggttc 1621tagaaggggc gaggctttaa gcgaggcaga atggtgggct ccagagtagg tgggtcttgg 1681attggtacca gagcctatgg aaagggtgtg gcttggaaca tttgggagac tgagcttgat 1741tctaaagggg acagatcttg agcaaggcaa gaagtgggat tcaggaatgg gccaagccag 1801ggttccagac agggtggggc ttagaatggg gcttccatgg tggtttcaga aagggcagcc 1861cctccccatg gtgcagtgaa gaaaatgttt tacaatggct gggtttgggc agtggagagg 1921ggacttggat aggagcttcc agatgggttt tgttaggggt gggggagaat ggctctggct 1981acgacttggg acggaagtgg cctgagaaga gtcgagtgat atggcttgta gggtgaggcg 2041tgggatccag agagaagcac cccaccacac acacccttcc ccactcccgt gatgaaacag 2101ctaggttaat aggaggacag aaccaacggg tctgtgggac tggcccaccc ctcttccccc 2161ttcccctgcg ccctccctcc ctccacacct ccacccgtcc tggggtggtt ggaggcctgg 2221tctggagccc ctatcctgca ccctctgcta tgtctgtgat gtcagtagtg cctgtgatcg 2281tgtgttgcca ttttgtctgg ctgtggcccc tccttctccc ctccagaccc ctaccctttc 2341ccaaaccctt cggtattgtt caaagaaccc ccctccccaa ggaagaacaa atatgattct 2401cctctcccaa ataaactcct taaccaccta gtcaaaaaaa aaaaaaaaa

By “NOGOA polypeptide” (or neurite outgrowth inhibitor A; neuriteoutgrowth inhibitor isoform A; human reticulon-4; human reticulon-4isoform A) is meant a polypeptide or fragment thereof having at leastabout 85% amino acid identity to NCBI Accession No. NP_065393.

1 medldqsplv sssdspprpq pafkyqfvre pedeeeeeee eeedededle elevlerkpa 61aglsaapvpt apaagaplmd fgndfvppap rgplpaappv aperqpswdp spvsstvpap 121splsaaavsp sklpeddepp arppppppas vspqaepvwt ppapapaapp stpaapkrrg 181ssgsvdetlf alpaasepvi rssaenmdlk eqpgntisag qedfpsvlle taaslpslsp 241lsaasfkehe ylgnlstvlp tegtlqenvs easkevseka ktllidrdlt efseleysem 301gssfsvspka esavivanpr eeiivknkde eeklvsnnil hnqqelptal tklvkedevv 361ssekakdsfn ekrvaveapm reeyadfkpf ervwevkdsk edsdmlaagg kiesnleskv 421dkkcfadsle qtnhekdses snddtsfpst pegikdrsga yitcapfnpa atesiatnif 481pllgdptsen ktdekkieek kaqivteknt stktsnpflv aaqdsetdyv ttdnltkvte 541evvanmpegl tpdlvqeace selnevtgtk iayetkmdlv qtsevmqesl ypaaqlcpsf 601eeseatpspv lpdivmeapl nsavpsagas viqpsssple assvnyesik hepenpppye 661eamsvslkkv sgikeeikep eninaalqet eapyisiacd liketklsae papdfsdyse 721makveqpvpd hselvedssp dsepvdlfsd dsipdvpqkq detvmlvkes ltetsfesmi 781eyenkeklsa lppeggkpyl esfklsldnt kdtllpdevs tlskkekipl qmeelstavy 841snddlfiske aqiretetfs dsspieiide fptlissktd sfsklareyt dlevshksei 901anapdgagsl pctelphdls lkniqpkvee kisfsddfsk ngsatskvll lppdvsalat 961qaeiesivkp kvlvkeaekk lpsdtekedr spsaifsael sktsvvdlly wrdikktgvv 1021fgaslfllls ltvfsivsvt ayialallsv tisfriykgv iqaiqksdeg hpfraylese 1081vaiseelvqk ysnsalghvn ctikelrrlf lvddlvdslk favlmwvfty vgalfngltl 1141lilalislfs vpviyerhqa qidhylglan knvkdamaki qakipglkrk ae

By “NOGOA nucleic acid molecule” (or neurite outgrowth inhibitor A;neurite outgrowth inhibitor isoform A; human reticulon-4; humanreticulon-4 isoform A) is meant a polynucleotide encoding an NOGOApolypeptide. An exemplary NOGOA nucleic acid molecule (e.g., mRNA) isprovided at NCBI Accession No. NM_020532.

1 agtccctgcc ctcccctggg gagggtgagt cacgccaaac tgggcggaga gtccgctggc 61ctcactccta gctcatctgg gcggcggcgg caagtgggga cagggcgggt ggcgcatcac 121cggcgcggag gcaggaggag cagtctcatt gttccgggag ccgtcaccac agtaggtccc 181tcggctcagt cggcccagcc cctctcagtc ctccccaacc cccacaaccg cccgcggctc 241tgagacgcgg ccccggcggc ggcggcagca gctgcagcat catctccacc ctccagccat 301ggaagacctg gaccagtctc ctctggtctc gtcctcggac agcccacccc ggccgcagcc 361cgcgttcaag taccagttcg tgagggagcc cgaggacgag gaggaagaag aggaggagga 421agaggaggac gaggacgaag acctggagga gctggaggtg ctggagagga agcccgccgc 481cgggctgtcc gcggccccag tgcccaccgc ccctgccgcc ggcgcgcccc tgatggactt 541cggaaatgac ttcgtgccgc cggcgccccg gggacccctg ccggccgctc cccccgtcgc 601cccggagcgg cagccgtctt gggacccgag cccggtgtcg tcgaccgtgc ccgcgccatc 661cccgctgtct gctgccgcag tctcgccctc caagctccct gaggacgacg agcctccggc 721ccggcctccc cctcctcccc cggccagcgt gagcccccag gcagagcccg tgtggacccc 781gccagccccg gctcccgccg cgcccccctc caccccggcc gcgcccaagc gcaggggctc 841ctcgggctca gtggatgaga ccctttttgc tcttcctgct gcatctgagc ctgtgatacg 901ctcctctgca gaaaatatgg acttgaagga gcagccaggt aacactattt cggctggtca 961agaggatttc ccatctgtcc tgcttgaaac tgctgcttct cttccttctc tgtctcctct 1021ctcagccgct tctttcaaag aacatgaata ccttggtaat ttgtcaacag tattacccac 1081tgaaggaaca cttcaagaaa atgtcagtga agcttctaaa gaggtctcag agaaggcaaa 1141aactctactc atagatagag atttaacaga gttttcagaa ttagaatact cagaaatggg 1201atcatcgttc agtgtctctc caaaagcaga atctgccgta atagtagcaa atcctaggga 1261agaaataatc gtgaaaaata aagatgaaga agagaagtta gttagtaata acatccttca 1321taatcaacaa gagttaccta cagctcttac taaattggtt aaagaggatg aagttgtgtc 1381ttcagaaaaa gcaaaagaca gttttaatga aaagagagtt gcagtggaag ctcctatgag 1441ggaggaatat gcagacttca aaccatttga gcgagtatgg gaagtgaaag atagtaagga 1501agatagtgat atgttggctg ctggaggtaa aatcgagagc aacttggaaa gtaaagtgga 1561taaaaaatgt tttgcagata gccttgagca aactaatcac gaaaaagata gtgagagtag 1621taatgatgat acttctttcc ccagtacgcc agaaggtata aaggatcgtt caggagcata 1681tatcacatgt gctcccttta acccagcagc aactgagagc attgcaacaa acatttttcc 1741tttgttagga gatcctactt cagaaaataa gaccgatgaa aaaaaaatag aagaaaagaa 1801ggcccaaata gtaacagaga agaatactag caccaaaaca tcaaaccctt ttcttgtagc 1861agcacaggat tctgagacag attatgtcac aacagataat ttaacaaagg tgactgagga 1921agtcgtggca aacatgcctg aaggcctgac tccagattta gtacaggaag catgtgaaag 1981tgaattgaat gaagttactg gtacaaagat tgcttatgaa acaaaaatgg acttggttca 2041aacatcagaa gttatgcaag agtcactcta tcctgcagca cagctttgcc catcatttga 2101agagtcagaa gctactcctt caccagtttt gcctgacatt gttatggaag caccattgaa 2161ttctgcagtt cctagtgctg gtgcttccgt gatacagccc agctcatcac cattagaagc 2221ttcttcagtt aattatgaaa gcataaaaca tgagcctgaa aaccccccac catatgaaga 2281ggccatgagt gtatcactaa aaaaagtatc aggaataaag gaagaaatta aagagcctga 2341aaatattaat gcagctcttc aagaaacaga agctccttat atatctattg catgtgattt 2401aattaaagaa acaaagcttt ctgctgaacc agctccggat ttctctgatt attcagaaat 2461ggcaaaagtt gaacagccag tgcctgatca ttctgagcta gttgaagatt cctcacctga 2521ttctgaacca gttgacttat ttagtgatga ttcaatacct gacgttccac aaaaacaaga 2581tgaaactgtg atgcttgtga aagaaagtct cactgagact tcatttgagt caatgataga 2641atatgaaaat aaggaaaaac tcagtgcttt gccacctgag ggaggaaagc catatttgga 2701atcttttaag ctcagtttag ataacacaaa agataccctg ttacctgatg aagtttcaac 2761attgagcaaa aaggagaaaa ttcctttgca gatggaggag ctcagtactg cagtttattc 2821aaatgatgac ttatttattt ctaaggaagc acagataaga gaaactgaaa cgttttcaga 2881ttcatctcca attgaaatta tagatgagtt ccctacattg atcagttcta aaactgattc 2941attttctaaa ttagccaggg aatatactga cctagaagta tcccacaaaa gtgaaattgc 3001taatgccccg gatggagctg ggtcattgcc ttgcacagaa ttgccccatg acctttcttt 3061gaagaacata caacccaaag ttgaagagaa aatcagtttc tcagatgact tttctaaaaa 3121tgggtctgct acatcaaagg tgctcttatt gcctccagat gtttctgctt tggccactca 3181agcagagata gagagcatag ttaaacccaa agttcttgtg aaagaagctg agaaaaaact 3241tccttccgat acagaaaaag aggacagatc accatctgct atattttcag cagagctgag 3301taaaacttca gttgttgacc tcctgtactg gagagacatt aagaagactg gagtggtgtt 3361tggtgccagc ctattcctgc tgctttcatt gacagtattc agcattgtga gcgtaacagc 3421ctacattgcc ttggccctgc tctctgtgac catcagcttt aggatataca agggtgtgat 3481ccaagctatc cagaaatcag atgaaggcca cccattcagg gcatatctgg aatctgaagt 3541tgctatatct gaggagttgg ttcagaagta cagtaattct gctcttggtc atgtgaactg 3601cacgataaag gaactcaggc gcctcttctt agttgatgat ttagttgatt ctctgaagtt 3661tgcagtgttg atgtgggtat ttacctatgt tggtgccttg tttaatggtc tgacactact 3721gattttggct ctcatttcac tcttcagtgt tcctgttatt tatgaacggc atcaggcaca 3781gatagatcat tatctaggac ttgcaaataa gaatgttaaa gatgctatgg ctaaaatcca 3841agcaaaaatc cctggattga agcgcaaagc tgaatgaaaa cgcccaaaat aattagtagg 3901agttcatctt taaaggggat attcatttga ttatacgggg gagggtcagg gaagaacgaa 3961ccttgacgtt gcagtgcagt ttcacagatc gttgttagat ctttattttt agccatgcac 4021tgttgtgagg aaaaattacc tgtcttgact gccatgtgtt catcatctta agtattgtaa 4081gctgctatgt atggatttaa accgtaatca tatctttttc ctatctatct gaggcactgg 4141tggaataaaa aacctgtata ttttactttg ttgcagatag tcttgccgca tcttggcaag 4201ttgcagagat ggtggagcta gaaaaaaaaa aaaaaaagcc cttttcagtt tgtgcactgt 4261gtatggtccg tgtagattga tgcagatttt ctgaaatgaa atgtttgttt agacgagatc 4321ataccggtaa agcaggaatg acaaagcttg cttttctggt atgttctagg tgtattgtga 4381cttttactgt tatattaatt gccaatataa gtaaatatag attatatatg tatagtgttt 4441cacaaagctt agacctttac cttccagcca ccccacagtg cttgatattt cagagtcagt 4501cattggttat acatgtgtag ttccaaagca cataagctag aagaagaaat atttctagga 4561gcactaccat ctgttttcaa catgaaatgc cacacacata gaactccaac atcaatttca 4621ttgcacagac tgactgtagt taattttgtc acagaatcta tggactgaat ctaatgcttc 4681caaaaatgtt gtttgtttgc aaatatcaaa cattgttatg caagaaatta ttaattacaa 4741aatgaagatt tataccattg tggtttaagc tgtactgaac taaatctgtg gaatgcattg 4801tgaactgtaa aagcaaagta tcaataaagc ttatagactt aaaaaaaaaa aaaaaaaaaa 4861aaaaaaaaaa a

By “GFAP” (or Glial fibrillary acidic protein) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. P14136.

1 merrritsaa rrsyvssgem mvgglapgrr lgpgtrlsla rmppplptrv dfslagalna 61gfketraser aemmelndrf asyiekvrfl eqqnkalaae lnqlrakept kladvyqael 121relrlrldql tansarleve rdnlaqdlat vrqklqdetn lrleaennla ayrqeadeat 181larldlerki esleeeirfl rkiheeevre lqeqlarqqv hveldvakpd ltaalkeirt 241qyeamassnm heaeewyrsk fadltdaaar naellrqakh eandyrrqlq sltcdleslr 301gtneslerqm reqeerhvre aasyqealar leeegqslkd emarhlqeyq dllnvklald 361ieiatyrkll egeenritip vqtfsnlqir etsldtksys eghlkrnivv ktvemrdgev 421ikeskqehkd vm

By “GFAP nucleic acid molecule” (or Glial fibrillary acidic protein) ismeant a polynucleotide encoding an GFAP polypeptide. An exemplary GFAPnucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No.NM_002055.

1 atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga gcagagccag 61agcaggatgg agaggagacg catcacctcc gctgctcgcc gctcctacgt ctcctcaggg 121gagatgatgg tggggggcct ggctcctggc cgccgtctgg gtcctggcac ccgcctctcc 181ctggctcgaa tgccccctcc actcccgacc cgggtggatt tctccctggc tggggcactc 241aatgctggct tcaaggagac ccgggccagt gagcgggcag agatgatgga gctcaatgac 301cgctttgcca gctacatcga gaaggttcgc ttcctggaac agcaaaacaa ggcgctggct 361gctgagctga accagctgcg ggccaaggag cccaccaagc tggcagacgt ctaccaggct 421gagctgcgag agctgcggct gcggctcgat caactcaccg ccaacagcgc ccggctggag 481gttgagaggg acaatctggc acaggacctg gccactgtga ggcagaagct ccaggatgaa 541accaacctga ggctggaagc cgagaacaac ctggctgcct atagacagga agcagatgaa 601gccaccctgg cccgtctgga tctggagagg aagattgagt cgctggagga ggagatccgg 661ttcttgagga agatccacga ggaggaggtt cgggaactcc aggagcagct ggcccgacag 721caggtccatg tggagcttga cgtggccaag ccagacctca ccgcagccct gaaagagatc 781cgcacgcagt atgaggcaat ggcgtccagc aacatgcatg aagccgaaga gtggtaccgc 841tccaagtttg cagacctgac agacgctgct gcccgcaacg cggagctgct ccgccaggcc 901aagcacgaag ccaacgacta ccggcgccag ttgcagtcct tgacctgcga cctggagtct 961ctgcgcggca cgaacgagtc cctggagagg cagatgcgcg agcaggagga gcggcacgtg 1021cgggaggcgg ccagttatca ggaggcgctg gcgcggctgg aggaagaggg gcagagcctc 1081aaggacgaga tggcccgcca cttgcaggag taccaggacc tgctcaatgt caagctggcc 1141ctggacatcg agatcgccac ctacaggaag ctgctagagg gcgaggagaa ccggatcacc 1201attcccgtgc agaccttctc caacctgcag attcgagaaa ccagcctgga caccaagtct 1261gtgtcagaag gccacctcaa gaggaacatc gtggtgaaga ccgtggagat gcgggatgga 1321gaggtcatta aggagtccaa gcaggagcac aaggatgtga tgtgaggcag gacccacctg 1381gtggcctctg ccccgtctca tgaggggccc gagcagaagc aggatagttg ctccgcctct 1441gctggcacat ttccccagac ctgagctccc caccacccca gctgctcccc tccctcctct 1501gtccctaggt cagcttgctg ccctaggctc cgtcagtatc aggcctgcca gacggcaccc 1561acccagcacc cagcaactcc aactaacaag aaactcaccc ccaaggggca gtctggaggg 1621gcatggccag cagcttgcgt tagaatgagg aggaaggaga gaaggggagg agggcggggg 1681gcacctacta catcgccctc cacatccctg attcctgttg ttatggaaac tgttgccaga 1741gatggaggtt ctctcggagt atctgggaac tgtgcctttg agtttcctca ggctgctgga 1801ggaaaactga gactcagaca ggaaagggaa ggccccacag acaaggtagc cctggccaga 1861ggcttgtttt gtcttttggt ttttatgagg tgggatatcc ctatgctgcc taggctgacc 1921ttgaactcct gggctcaagc agtctaccca cctcagcctc ctgtgtagct gggattatag 1981attggagcca ccatgcccag ctcagagggt tgttctccta gactgaccct gatcagtcta 2041agatgggtgg ggacgtcctg ccacctgggg cagtcacctg cccagatccc agaaggacct 2101cctgagcgat gactcaagtg tctcagtcca cctgagctgc catccaggga tgccatctgt 2161gggcacgctg tgggcaggtg ggagcttgat tctcagcact tgggggatct gttgtgtacg 2221tggagaggga tgaggtgctg ggagggatag aggggggctg cctggccccc agctgtgggt 2281acagagaggt caagcccagg aggactgccc cgtgcagact ggaggggacg ctggtagaga 2341tggaggagga ggcaattggg atggcgctag gcatacaagt aggggttgtg ggtgaccagt 2401tgcacttggc ctctggattg tgggaattaa ggaagtgact catcctcttg aagatgctga 2461aacaggagag aaaggggatg tatccatggg ggcagggcat gactttgtcc catttctaaa 2521ggcctcttcc ttgctgtgtc ataccaggcc gccccagcct ctgagcccct gggactgctg 2581cttcttaacc ccagtaagcc actgccacac gtctgaccct ctccacccca tagtgaccgg 2641ctgcttttcc ctaagccaag ggcctcttgc ggtcccttct tactcacaca caaaatgtac 2701ccagtattct aggtagtgcc ctattttaca attgtaaaac tgaggcacga gcaaagtgaa 2761gacactggct catattcctg cagcctggag gccgggtgct cagggctgac acgtccaccc 2821cagtgcaccc actctgcttt gactgagcag actggtgagc agactggtgg gatctgtgcc 2881cagagatggg actgggaggg cccacttcag ggttctcctc tcccctctaa ggccgaagaa 2941gggtccttcc ctctccccaa gacttggtgt cctttccctc cactccttcc tgccacctgc 3001tgctgctgct gctgctaatc ttcagggcac tgctgctgcc tttagtcgct gaggaaaaat 3061aaagacaaat gctgcgccct tccccaaaaa aaaaaaa

By “s100b” (or S-100 protein beta chain; S-100 protein subunit beta;S100 calcium-binding protein B) is meant a polypeptide or fragmentthereof having at least about 85% amino acid identity to NCBI AccessionNo. P04271.

1 mselekamva lidvfhqysg regdkhklkk selkelinne lshfleeike qevvdkvmet 61ldndgdgecd fqefmafvam vttacheffe he

By “s100b nucleic acid molecule” (or S-100 protein beta chain; S-100protein subunit beta; S100 calcium-binding protein B) is meant apolynucleotide encoding an s100b polypeptide. An exemplary s100b nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006272.

1 gggcagaggg aataagaggc tgcctctgcc caccagtcct gccgcccagg acccgcagca 61gagacgacgc ctgcagcaag gagaccagga aggggtgaga caaggaagag gatgtctgag 121ctggagaagg ccatggtggc cctcatcgac gttttccacc aatattctgg aagggaggga 181gacaagcaca agctgaagaa atccgaactg aaggagctca tcaacaatga gctttcccat 241ttcttagagg aaatcaaaga gcaggaggtt gtggacaaag tcatggaaac actggacaat 301gatggagacg gcgaatgtga cttccaggaa ttcatggcct ttgttgccat ggttactact 361gcctgccacg agttctttga acatgagtga gattagaaag cagccaaacc tttcctgtaa 421cagagacggt catgcaagaa agcagacagc aagggcttgc agcctagtag gagctgagct 481ttccagccgt gttgtagcta attaggaagc ttgatttgct ttgtgattga aaaattgaaa 541acctctttcc aaaggctgtt ttaacggcct gcatcattct ttctgctata ttaggcctgt 601gtgtaagctg actggcccca gggactcttg ttaacagtaa cttaggagtc aggtctcagt 661gataaagcgt gcaccgtgca gcccgccatg gccgtgtaga ccctaacccg gagggaaccc 721tgactacaga aattaccccg gggcaccctt aaaacttcca ctacctttaa aaaacaaagc 781cttatccagc attatttgaa aacactgctg ttctttaaat gcgttcctca tccatgcaga 841taacagctgg ttggccggtg tggccctgca agggcgtggt ggcttcggcc tgcttcccgg 901gatgcgcctg atcaccaggt gaacgctcag cgctggcagc gctcctggaa aaagcaactc 961catcagaact cgcaatccga gccagctctg ggggctccag cgtggcctcc gtgacccatg 1021cgattcaagt cgcggctgca ggatccttgc ctccaacgtg cctccagcac atgcggcttc 1081cgagggcact accgggggct ctgagccacc gcgagggcct gcgttcaata aaaag

By “PAX6 polypeptide” (or paired box protein PAX6) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. AAK95849.1.

MQNSHSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDISRILQVSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPEVVSKIAQYKRECPSIFAWEIRDRLLSEGVCTNDNIPSVSSINRVLRNLASEKQQMGADGMYDKLRMLNGQTGSWGTRPGWYPGTSVPGQPTQDGCQQQEGGGENTNSISSNGEDSDEAQMRLQLKRKLQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQVWFSNRRAKWRREEKLRNQRRQASNTPSHIPISSSFSTSVYQPIPQPTTPVSSFTSGSMLGRTDTALTNTYSALPPMPSFTMANNLPMQPPVPSQTSSYSCMLPTSPSVNGRSYDTYTPPHMQTHMNSQPMGTSGTTSTGLISPGVSVPVQVPGSEPDMSQYWPRLQ

By “PAX6 polynucleotide” (or paired box protein PAX6) is meant apolynucleotide encoding an PAX6 polypeptide. An exemplary PAX6 nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. AY047583.

1 agggggaaga ctttaactag gggcgcgcag atgtgtgagg ccttttattg tgagagtgga 61cagacatccg agatttcaga gccccatatt cgagccccgt ggaatcccgc ggcccccagc 121cagagccagc atgcagaaca gtcacagcgg agtgaatcag ctcggtggtg tctttgtcaa 181cgggcggcca ctgccggact ccacccggca gaagattgta gagctagctc acagcggggc 241ccggccgtgc gacatttccc gaattctgca ggtgtccaac ggatgtgtga gtaaaattct 301gggcaggtat tacgagactg gctccatcag acccagggca atcggtggta gtaaaccgag 361agtagcgact ccagaagttg taagcaaaat agcccagtat aagcgggagt gcccgtccat 421ctttgcttgg gaaatccgag acagattact gtccgagggg gtctgtacca acgataacat 481accaagcgtg tcatcaataa acagagttct tcgcaacctg gctagcgaaa agcaacagat 541gggcgcagac ggcatgtatg ataaactaag gatgttgaac gggcagaccg gaagctgggg 601cacccgccct ggttggtatc cggggacttc ggtgccaggg caacctacgc aagatggctg 661ccagcaacag gaaggagggg gagagaatac caactccatc agttccaacg gagaagattc 721agatgaggct caaatgcgac ttcagctgaa gcggaagctg caaagaaata gaacatcctt 781tacccaagag caaattgagg ccctggagaa agagtttgag agaacccatt atccagatgt 841gtttgcccga gaaagactag cagccaaaat agatctacct gaagcaagaa tacaggtatg 901gttttctaat cgaagggcca aatggagaag agaagaaaaa ctgaggaatc agagaagaca 961ggccagcaac acacctagtc atattcctat cagcagtagt ttcagcacca gtgtctacca 1021accaattcca caacccacca caccggtttc ctccttcaca tctggctcca tgttgggccg 1081aacagacaca gccctcacaa acacctacag cgctctgccg cctatgccca gcttcaccat 1141ggcaaataac ctgcctatgc aacccccagt ccccagccag acctcctcat actcctgcat 1201gctgcccacc agcccttcgg tgaatgggcg gagttatgat acctacaccc ccccacatat 1261gcagacacac atgaacagtc agccaatggg cacctcgggc accacttcaa caggactcat 1321ttcccctggt gtgtcagttc cagttcaagt tcccggaagt gaacctgata tgtctcaata 1381ctggccaaga ttacagtaa

By “Nestin polypeptide” is meant a polypeptide or fragment thereofhaving at least about 85% amino acid identity to NCBI Accession No.NP_006608.1.

MEGCMGEESFQMWELNRRLEAYLARVKALEEQNELLSAELGGLRAQSADTSWRAHADDELAALRALVDQRWREKHAAEVARDNLAEELEGVAGRCQQLRLARERTTEEVARNRRAVEAEKCARAWLSSQVAELERELEALRVAHEEERVGLNAQAACAPRCPAPPRGPPAPAPEVEELARRLGEAWRGAVRGYQERVAHMETSLGQARERLGRAVQGAREGRLELQQLQAERGGLLERRAALEQRLEGRWQERLRATEKFQLAVEALEQEKQGLQSQIAQVLEGRQQLAHLKMSLSLEVATYRTLLEAENSRLQTPGGGSKTSLSFQDPKLELQFPRTPEGRRLGSLLPVLSPTSLPSPLPATLETPVPAFLKNQEFLQARTPTLASTPIPPTPQAPSPAVDAEIRAQDAPLSLLQTQGGRKQAPEPLRAEARVAIPASVLPGPEEPGGQRQEASTGQSPEDHASLAPPLSPDHSSLEAKDGESGGSRVFSICRGEGEGQIWGLVEKETAIEGKVVSSLQQEIWEEEDLNRKEIQDSQVPLEKETLKSLGEEIQESLKTLENQSHETLERENQECPRSLEEDLETLKSLEKENKELLKDVEVVRPLEKEAVGQLKPTGKEDTQTLQSLQKENQELMKSLEGNLETFLFPGTENQELVSSLQENLESLTALEKENQEPLRSPEVGDEEALRPLTKENQEPLRSLEDENKEAFRSLEKENQEPLKTLEEEDQSIVRPLETENHKSLRSLEEQDQETLRTLEKETQQRRRSLGEQDQMTLRPPEKVDLEPLKSLDQEIARPLENENQEFLKSLKEESVEAVKSLETEILESLKSAGQENLETLKSPETQAPLWTPEEINQGAMNPLEKEIQEPLESVEVNQETFRLLEEENQESLRSLGAWNLENLRSPEEVDKESQRNLEEEENLGKGEYQESLRSLEEEGQELPQSADVQRWEDTVEKDQELAQESPPGMAGVENEDEAELNLREQDGFTGKEEVVEQGELNATEEVWIPGEGHPESPEPKEQRGLVEGASVKGGAEGLQDPEGQSQQVGAPGLQAPQGLPEAIEPLVEDDVAPGGDQASPEVMLGSEPAMGESAAGAEPGPGQGVGGLGDPGHLTREEVMEPPLEEESLEAKRVQGLEGPRKDLEEAGGLGTEFSELPGKSRDPWEPPREGREESEAEAPRGAEEAFPAETLGHTGSDAPSPWPLGSEEAEEDVPPVLVSPSPTYTPILEDAPGPQPQAEGSQEASWGVQGRAEALGKVESEQEELGSGEIPEGPQEEGEESREESEEDELGETLPDSTPLGFYLRSPTSPRWDPTGEQRPPPQGETGKEGWDPAVLASEGLEAPPSEKEEGEEGEEECGRDSDLSEEFEDLGTEAPFLPGVPGEVAEPLGQVPQLLLDPAAWDRDGESDGFADEEESGEEGEEDQEEGREPGAGRWGPGSSVGSLQALSSSQRGEFLESDSVSVSVPWDDSLRGAVAGAPKTALETESQDSAEPSGSEEESDPVSLEREDKVPGPLEIPSGMEDAGPGADIIGVNGQGPNLEGKSQHVNGGVMNGLEQSEEVGQGMPLVSEGDRGSPFQEEEGSALKTSWAGAPVHLGQ GQFLKFTQREGDRESWSSGED

By “Nestin polynucleotide” is meant a polynucleotide encoding an Nestinpolypeptide. An exemplary Nestin nucleic acid molecule (e.g., mRNA) isprovided at NCBI Accession No. NM_006617.

1 gctactccca ccccgccccg ccccgtcatt gtccccgtcg gtctcttttc tcttccgtcc 61taaaagctct gcgagccgct cccttctccc ggtgccccgc gtctgtccat cctcagtggg 121tcagacgagc aggatggagg gctgcatggg ggaggagtcg tttcagatgt gggagctcaa 181tcggcgcctg gaggcctacc tggcccgggt caaggcgctg gaggagcaga atgagctgct 241cagcgcggag ctcggggggc tccgggcaca atccgcggac acctcctggc gggcgcatgc 301cgacgacgag ctggcggccc tgcgggccct cgttgaccaa cgctggcggg agaagcacgc 361ggccgaggtg gcgcgcgaca acctggctga agagctggag ggcgtggcag gccgatgcca 421gcagctgcgg ctggcccggg agcggacgac ggaggaggta gcccgcaacc ggcgcgccgt 481cgaggcagag aaatgcgccc gggcctggct gagtagccag gtggcagagc tggagcgcga 541gctagaggct ctacgcgtgg cgcacgagga ggagcgcgtc ggcctgaacg cgcaggctgc 601ctgtgccccc cgctgccccg cgccgccccg cgggcctccc gcgccggccc cggaggtaga 661ggagctggca aggcgactgg gcgaggcgtg gcgcggggca gtgcgcggct accaggagcg 721cgtggcacac atggagacgt cgctgggcca ggcccgcgag cggctgggcc gggcggtgca 781gggtgcccgc gagggccgcc tggagctgca gcagctccag gctgagcgcg gaggcctcct 841ggagcgcagg gcagcgttgg aacagaggtt ggagggccgc tggcaggagc ggctgcgggc 901tactgaaaag ttccagctgg ctgtggaggc cctggagcag gagaaacagg gcctacagag 961ccagatcgct caggtcctgg aaggtcggca gcagctggcg cacctcaaga tgtccctcag 1021cctggaggtg gccacgtaca ggaccctcct ggaggctgag aactcccggc tgcaaacacc 1081tggcggtggc tccaagactt ccctcagctt tcaggacccc aagctggagc tgcaattccc 1141taggacccca gagggccggc gtcttggatc tttgctccca gtcctgagcc caacttccct 1201cccctcaccc ttgcctgcta cccttgagac acctgtgcca gcctttctta agaaccaaga 1261attcctccag gcccgtaccc ctaccttggc cagcaccccc atccccccca cacctcaggc 1321accctctcct gctgtagatg cagagatcag agcccaggat gctcctctct ctctgctcca 1381gacacagggt gggaggaaac aggctccaga gcccctgcgg gctgaagcca gggtggccat 1441tcctgccagc gtcctgcctg gaccagagga gcctgggggc cagcggcaag aggccagtac 1501aggccagtcc ccagaggacc atgcctcctt ggcaccaccc ctcagccctg accactccag 1561tttagaggct aaggatggag aatccggtgg gtctagagtg ttcagcatat gccgagggga 1621aggtgaaggg caaatctggg ggttggtaga gaaagaaaca gccatagagg gcaaagtggt 1681aagcagcttg cagcaggaaa tatgggaaga agaggatcta aacaggaagg aaatccagga 1741ctcccaggtt cctttggaaa aagaaaccct gaagtctctg ggagaggaga ttcaagagtc 1801actgaagact ctggaaaacc agagccatga gacactagaa agggagaatc aagaatgtcc 1861gaggtcttta gaagaagact tagaaacact aaaaagtcta gaaaaggaaa ataaagagct 1921attaaaggat gtggaggtag tgagacctct agaaaaagag gctgtaggcc aacttaagcc 1981tacaggaaaa gaggacacac agacattgca atccctgcaa aaggagaatc aagaactaat 2041gaaatctctt gaaggtaatc tagagacatt tttatttcca ggaacggaaa atcaagaatt 2101agtaagttct ctgcaagaga acttagagtc attgacagct ctggaaaagg agaatcaaga 2161gccactgaga tctccagaag taggggatga ggaggcactg agacctctga caaaggagaa 2221tcaggaaccc ctgaggtctc ttgaagatga gaacaaagag gcctttagat ctctagaaaa 2281agagaaccag gagccactga agactctaga agaagaggac cagagtattg tgagacctct 2341agaaacagag aatcacaaat cactgaggtc tttagaagaa caggaccaag agacattgag 2401aactcttgaa aaagagactc aacagcgacg gaggtctcta ggggaacagg atcagatgac 2461attaagaccc ccagaaaaag tggatctaga accactgaag tctcttgacc aggagatagc 2521tagacctctt gaaaatgaga atcaagagtt cttaaagtca ctcaaagaag agagcgtaga 2581ggcagtaaaa tctttagaaa cagagatcct agaatcactg aagtctgcgg gacaagagaa 2641cctggaaaca ctgaaatctc cagaaactca agcaccactg tggactccag aagaaataaa 2701tcagggggca atgaatcctc tagaaaagga aattcaagaa ccactggagt ctgtggaagt 2761gaaccaagag acattcagac tcctggaaga ggagaatcag gaatcattga gatctctggg 2821agcatggaac ctggagaatt tgagatctcc agaggaggta gacaaggaaa gtcaaaggaa 2881tctggaagag gaagagaacc tgggaaaggg agagtaccaa gagtcactga ggtctctgga 2941ggaggaggga caggagctgc cgcagtctgc agatgtgcag aggtgggaag atacggtgga 3001gaaggaccaa gaactggctc aggaaagccc tcctgggatg gctggagtgg aaaatgagga 3061tgaggcagag ctgaatctga gggagcagga tggcttcact gggaaggagg aggtggtaga 3121gcagggagag ctgaatgcca cagaggaggt ctggatccca ggcgaggggc acccagagag 3181ccctgagccc aaagagcaga gaggcctggt tgagggagcc agtgtgaagg gaggggctga 3241gggcctccag gaccctgaag ggcaatcaca acaggtgggg gccccaggcc tccaggctcc 3301ccaggggctg ccagaggcga tagagcccct ggtggaagat gatgtggccc cagggggtga 3361ccaagcctcc ccagaggtca tgttggggtc agagcctgcc atgggtgagt ctgctgcggg 3421agctgagcca ggcccggggc agggggtggg agggctgggg gacccaggcc atctgaccag 3481ggaagaggtg atggaaccac ccctggaaga ggagagtttg gaggcaaaga gggttcaggg 3541cttggaaggg cctagaaagg acctagagga ggcaggtggt ctggggacag agttctccga 3601gctgcctggg aagagcagag acccttggga gcctcccagg gagggtaggg aggagtcaga 3661ggctgaggcc cccaggggag cagaggaggc gttccctgct gagaccctgg gccacactgg 3721aagtgatgcc ccttcacctt ggcctctggg gtcagaggaa gctgaggagg atgtaccacc 3781agtgctggtc tcccccagcc caacgtacac cccgatcctg gaagatgccc ctgggcctca 3841gcctcaggct gaagggagtc aggaggctag ctggggggtg caggggaggg ctgaagccct 3901ggggaaagta gagagcgagc aggaggagtt gggttctggg gagatccccg agggccccca 3961ggaggaaggg gaggagagca gagaagagag cgaggaggat gagctcgggg agacccttcc 4021agactccact cccctgggct tctacctcag gtcccccacc tcccccaggt gggaccccac 4081tggagagcag aggccacccc ctcaagggga gactggaaag gagggctggg atcctgctgt 4141cctggcttcc gagggccttg aggccccacc ctcagaaaag gaggaggggg aggagggaga 4201agaggagtgt ggccgtgact ctgacctgtc agaagaattt gaggacctgg ggactgaggc 4261accttttctt cctggggtcc ctggggaggt ggcagaacct ctgggccagg tgccccagct 4321gctactggat cctgcagcct gggatcgaga tggggagtcc gatgggtttg cagatgagga 4381agaaagtggg gaggagggag aggaggatca ggaggagggg agggagccag gggctgggcg 4441gtgggggcca gggtcttctg ttggcagcct ccaggccctg agtagctccc agagagggga 4501attcctggag tctgattctg tgagtgtcag tgtcccctgg gatgacagct tgaggggtgc 4561agtggctggt gcccccaaga ctgccctgga aacggagtcc caggacagtg ctgagccttc 4621tggctcagag gaagagtctg accctgtttc cttggagagg gaggacaaag tccctggccc 4681tctagagatc cccagtggga tggaggatgc aggcccaggg gcagacatca ttggtgttaa 4741tggccagggt cccaacttgg aggggaagtc acagcatgtg aatgggggag tgatgaacgg 4801gctggagcag tctgaggaag tggggcaagg aatgccgcta gtctctgagg gagaccgagg 4861gagccccttt caggaggagg aggggagtgc tctgaagacc tcttgggcag gggctcctgt 4921tcacctgggc cagggtcagt tcctgaagtt cactcagagg gaaggagata gagagtcctg 4981gtcctcaggg gaggactagg aaaagaccat ctgcccggca ctggggactt aggggtgcgg 5041ggaggggaag gacgcctcca agcccgctcc ctgctcagga gcagcactct taacttacga 5101tctcttgaca tatggtttct ggctgagagg cctggcccgc taaggtgaaa aggggtgtgg 5161caaaggagcc tactccaaga atggaggctg taggaatata acctcccacc ctgcaaaggg 5221aatctcttgc ctgctccatc tcataggcta agtcagctga atcccgatag tactaggtcc 5281ccttccctcc gcatcccgtc agctggaaaa ggcctgtggc ccagaggctt ctccaaaggg 5341agggtgacat gctggctttt gtgcccaagc tcaccagccc tgcgccacct cactgcagta 5401gtgcaccatc tcactgcagt agcacgccct cctgggccgt ctggcctgtg gctaatggag 5461gtgacggcac tcccatgtgc tgactccccc catccctgcc acgctgtggc cctgcctggc 5521tagtccctgc ctgaataaag taatgcctcc gcttcaaaaa aaaaaaaaaa aaaaaaaaaa 5581aaaaaaaaaa a

By “LHX6 polypeptide” (or LIM homeobox 6) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. AAI03937.1.

MAQPGSGCKATTRCLEGTAPPAMAQSDAEALAGALDKDEGQASPCTPSTPSVCSPPSAASSVPSAGKNICSSCGLEILDRYLLKVNNLIWHVRCLECSVCRTSLRQQNSCYIKNKEIFCKMDYFSRFGTKCARCGRQIYASDWVRRARGNAYHLACFACFSCKRQLSTGEEFGLVEEKVLCRIHYDTMIENLKRAAENGNGLTLEGAVPSEQDSQPKPAKRARTSFTAEQLQVMQAQFAQDNNPDAQTLQKLADMTGLSRRVIQVWFQNCRARHKKHTPQHPVPPSGAPPSRLPSALSDDIHYTPFSSPERARMVTLHGYIESHPFSVLTLPALPHLPVGAPQLPLSR

By “LHX6 polynucleotide” (or LIM homeobox 6) is meant a polynucleotideencoding an LHX6 polypeptide. An exemplary LHX6 nucleic acid molecule(e.g., mRNA) is provided at NCBI Accession No. BC103936.

1 cccgccaccg accaggtgat ggcccagcca gggtccggct gcaaagcgac cacccgctgt 61cttgaaggga ccgcgccgcc cgccatggct cagtctgacg ccgaggccct ggcaggagct 121ctggacaagg acgagggtca ggcctcccca tgtacgccca gcacgccatc tgtctgctca 181ccgccctctg ccgcctcctc cgtgccgtct gcaggcaaga acatctgctc cagctgcggc 241ctcgagatcc tggaccgata tctgctcaag gtcaacaacc tcatctggca cgtgcggtgc 301ctcgagtgct ccgtgtgtcg cacgtcgctg aggcagcaga acagctgcta catcaagaac 361aaggagatct tctgcaagat ggactacttc agccgattcg ggaccaagtg tgcccggtgc 421ggccgacaga tctacgccag cgactgggtg cggagagctc gcggcaacgc ctaccacctg 481gcctgcttcg cctgcttctc gtgcaagcgc cagctgtcca ctggtgagga gttcggcctg 541gtcgaggaga aggtgctctg ccgcatccac tacgacacca tgattgagaa cctcaagagg 601gccgccgaga acgggaacgg cctcacgttg gagggggcag tgccctcgga acaggacagt 661caacccaagc cggccaagcg cgcgcggacg tccttcaccg cggaacagct gcaggttatg 721caggcgcagt tcgcgcagga caacaacccc gacgctcaga cgctgcagaa gctggcggac 781atgacgggcc tcagccggag agtcatccag gtgtggtttc aaaactgccg ggcgcgtcat 841aaaaagcaca cgccgcaaca cccagtgccg ccctcggggg cgcccccgtc ccgccttccc 901tccgccctgt ccgacgacat ccactacacc ccgttcagca gccccgagcg ggcgcgcatg 961gtcaccctgc acggctacat tgagagtcat cctttttcag tactaacgct gccggcactt 1021ccgcatctgc ccgtgggcgc cccacagctg cccctcagcc gctgagatcc agtgtccaag 1081ctgcggccag gagtccaccc acctccgcat ccacccccgt ccgccatcct gcccaccacc 1141aggtcggttc ccgaggcctg gcctttccct ctcctgctga gaaccagaac ccaccaggag 1201caccacagag tcctcctctt ggaaggcaga actccctgaa atctggaatc agggtggaaa 1261cagcctgttt ttcccattta aacaggagtc ctcttcaact tcagctgatt acaataacaa 1321aaggcggaat tgaattgtgc gatgccaacg gccttctcat ttacaggttt ttttccccca 1381cattggcctt tatttactac ttccttggaa ccatctctga attctgaata gctgacaacc 1441cccaatgtta tccactctgt tgcttttgtc tggaaaactc tacagtgttt gtgggatgtc 1501cccaaaggta agctatgttc taattttatc atttccatct gtctggttat gtcaagttaa 1561ttcagaaaga gaagagacag tgaccaaccc tgagaggcct aatagggcag agatggaggc 1621ctgcccagac taggaggcag cggggataga cagggaatgg ggagaagaaa gacccccatt 1681ggtttggaaa tcaaggagag ggcggtgaca tattggacca gaagaggcac tagccatttt 1741aaggagagga aagagaaaac tctggggtca gggagagacc ctacccccac ctaattatcc 1801agcatatatg taagaaacat agcagcgatg gtattcgatc tgtgccatga ctcttctgaa 1861tgtttggaca ggttagagtt ggggacccct gttggccact tgttgacctc tcatagtggt 1921gcttgggcca ggtcttctca atggaagggg aatcccttat aggggagagg gaacagagcc 1981cagtgaaatg gcagtcagaa tgttaaccct ggatccatct ctaagtagag agagggtgcc 2041cattgcctag gtgagtgtgc caagctcagg attccaactg gtgcctctga gcttcccaat 2101caatacttcc tggagccagc cccacccacc cctgagaaca gaggtcagac acagctgcgt 2161aacatccatc ctgctacaac tcttccaccc caaacaaaag ggctcaggct acacacgacc 2221atgatttatg ttttcagggg atgcccattt gtcccaagct tatcctgtaa ttctagaatt 2281acctggtgtc ctgatgcatt ttccactaga ggttgctaat cagcatgttt tagcccaagt 2341ccaccttcct gctgtggtta acctgttatg ttgcttttgg aaggagactc taagacaggg 2401aaagcaagtt catggtacat acgcagccat tgtctctgtt tttacccatg gcagacattg 2461ctaatcaatg gcagctctat ttcactgagt ctggataagg tttcagagtt caaatgcttg 2521acgttggcac ttaacatgaa agcctatagg tcattcttgc tctgggatct acaggcaggg 2581taggcacagg tgcagcctaa gaagggaacc tgcttcctct cccttccaaa gacagtgaca 2641gctgactgag ggcaaagagc aggcaccact cagaacgtgg tgagtacagc tcagctcagc 2701actcagtcag tggtaacttg tgcccagccc tgtgctaggc gctgacatta acaggagcaa 2761ccagggccca attcctggcc ttggagctca aatctttcct ttgatttttg ctcctgatca 2821tcaaggcccc agtgg

By “LHX8 polypeptide” (or LIM homeobox 8) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. AAH40321.1.

MQILSRCQGLMSEECGRTTALAAGRTRKGAGEEGLVSPEGAGDEDSCSSSAPLSPSSSPRSMASGSGCPPGKCVCNSCGLEIVDKYLLKVNDLCWHVRCLSCSVCRTSLGRHTSCYIKDKDIFCKLDYFRRYGTRCSRCGRHIHSTDWVRRAKGNVYHLACFACFSCKRQLSTGEEFALVEEKVLCRVHYDCMLDNLKREVENGNGISVEGALLTEQDVNHPKPAKRARTSFTADQLQVMQAQFAQDNNPDAQTLQKLAERTGLSRRVIQVWFQNCRARHKKHVSPNHSSSTPVTAAPPSRLSPPMLEEMAYSAYVPQDGTMLTALHSYMDAHSPTTLGLQPLLPHSMTQ LPISHT

By “LHX8 polynucleotide” (or LIM homeobox 8) is meant a polynucleotideencoding an LHX8 polypeptide. An exemplary LHX8 nucleic acid molecule(e.g., mRNA) is provided at NCBI Accession No. BC040321.

1 agcggcaaga ggctagcggc tggaccactt gtgctggagt ggtaaagaac tatcatgaat 61ccatttactg aaagtgtcca tttctgaact caccctaaag aggacaaaca ccgcaaagta 121gttaaaagtc aggcattcgc gtcggacgtc tgggtttgaa ttctgccctg gcttgactgg 181aaacgcttcc cctatttctt ccgtagcgga ccgggagagc ttactggcgc tctgcgaacc 241ggctggaaag aaacaccgag tcactcgtac agactcttgg tcgcagaact tggctttccg 301ctattggtcc tccagaaccg cttgaaacaa ctggccccag ctggcgcatc agaccgcagt 361gaggaatgcc gcggggcggg tggcgaaggc agggtctgcc cgccagtgga ttcccgggtg 421tcccgcgtgg agcaggcttg cccagctggg aagcccatca aacctcagtc ttggcccaca 481gtgggagaga gaccagtggg tcccagacgg aggccatcgc ccgcttttgg cgacctccac 541tggcgtgaat aaaagcaccc ctctcttacc ctcagaaact gtgggtagca aggtataaaa 601cggagtctgg gaccggtaag tcccaaggtg agcccgtata cagctctgcc atctctgagg 661ggttatgcag attctgagca ggtgtcaggg gctcatgtca gaggagtgcg ggcggactac 721agccctggcg gccgggagga ctcgcaaagg cgccggggaa gagggactgg tgagccccga 781gggagcgggg gacgaggact cgtgctcctc ctcggccccg ctgtccccgt cgtcctcgcc 841ccggtccatg gcctcgggct ccggctgccc tcctggcaag tgtgtgtgca acagttgcgg 901cctggagatc gtggacaaat accttctcaa ggtgaatgac ctatgctggc atgtccggtg 961tctctcctgc agtgtttgca gaacctccct aggaaggcac accagctgtt atattaaaga 1021caaagacatt ttctgcaaac ttgattattt cagaaggtat ggaactcgct gctctcgatg 1081tgggagacac atccattcta ctgactgggt ccggagagcc aaggggaatg tctatcactt 1141ggcatgcttt gcctgctttt cctgcaaaag gcaactttcc acaggagagg agtttgcttt 1201ggtggaagag aaagtcctct gcagagtaca ttatgactgc atgctggata atttaaaaag 1261agaagtagaa aatgggaatg ggattagtgt ggaaggtgcc ctcctcacag agcaagatgt 1321taaccatcca aaaccagcaa aaagagctcg gaccagcttt acagcagatc agcttcaggt 1381tatgcaagca caatttgctc aggacaacaa cccagatgca cagacactcc agaaattggc 1441agaaaggaca ggcttgagca gacgtgtgat acaggtgtgg tttcagaatt gtagagcacg 1501ccacaagaaa cacgtcagtc ctaatcactc atcctccacc ccagtcacag cagccccacc 1561ctccaggctg tctccaccca tgttagaaga aatggcttat tctgcctacg tgccccaaga 1621tggaacgatg ttaactgcgc tgcatagtta tatggatgct cattcaccaa caactcttgg 1681actccagccc ttgttacccc attcaatgac acaactgcca ataagtcata cctaattctt 1741ttttcaggga tagacttgat taaggatata aatttgtcat ttattatgta taaaatacca 1801ttgaaaagat attactgtta attttttatt taacacctaa agcatttcca acatcacttt 1861gctgcccagg tatgtatcta tagttggcct gcaagacact tttattaatt cttcattttt 1921tgtaaaactt atgtttacaa gaagaaaaca aatcaaaaca ttttttgtat tgtctggaaa 1981tagttcactc tagtgtgtat ctgttaattt atttgtcatc aaaagagcac tttgcctaaa 2041agaaaggact gacaagtgtg caaaatgttt acaatctttt gtgaaattgt agtttatcat 2101tagtttgtat ctgtaagtta ttgtaataaa tattacctgt attttttgtt atatacaact 2161ttatactttg aagcttgtat ctgtgaattt gcaactgaaa tttattttgc caatgttttc 2221tgaatgaact gaataaagct tctgttgtag catgccatgc aaacacatta ttgtgtttgt 2281ggttgatgaa ttatggctgt aaataacact atagtttaat aagcccacca ttctgagttt 2341attaaacatt ttccattctt gtgaaaattt caaaaaaaaa aaaaaaaaaa aaagaaaaaa 2401aaaaaaaaaa a

By “TBR1 polypeptide” (or T-box, brain 1 (TBR1)) is meant a polypeptideor fragment thereof having at least about 85% amino acid identity toNCBI Accession No. NP_006584.1.

MQLEHCLSPSIMLSKKFLNVSSSYPHSGGSELVLHDHPIISTTDNLERSSPLKKITRGMTNQSDTDNFPDSKDSPGDVQRSKLSPVLDGVSELRHSFDGSAADRYLLSQSSQPQSAATAPSAMFPYPGQHGPAHPAFSIGSPSRYMAHHPVITNGAYNSLLSNSSPQGYPTAGYPYPQQYGHSYQGAPFYQFSSTQPGLVPGKAQVYLCNRPLWLKFHRHQTEMIITKQGRRMFPFLSFNISGLDPTAHYNIFVDVILADPNHWRFQGGKWVPCGKADTNVQGNRVYMHPDSPNTGAHWMRQEISFGKLKLTNNKGASNNNGQMVVLQSLHKYQPRLHVVEVNEDGTEDTSQPGRVQTFTFPETQFIAVTAYQNTDITQLKIDHNPFAKGFRDNYDTIYTGCDMDRLTPSPNDSPRSQIVPGARYAMAGSFLQDQFVSNYAKARFHPGAGAGPGPGTDRSVPHTNGLLSPQQAEDPGAPSPQRWFVTPANNRLDFAASAYDTATDFAGNAATLLSYAAAGVKALPLQAAGCTGRPLGYYADPSGWGARSPPQYCGTKSGSVLPCWPNSAAAAARMAGANPYLGEEAEGLAAERSPLPPGAAEDAKPKDLSDSSWIETPSSIKSIDSSDSGIYEQAKRRRISPADTPVSESSSPLKSEVLAQRDCEKNCAKDISGYYGFYSHS

By “TBR1 polynucleotide” (or T-box, brain 1 (TBR1)) is meant apolynucleotide encoding an TBR1 polypeptide. An exemplary TBR1 nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. NM_006593.

1 gtcgctacca ggagccaggt gattatccta attaatgtct atctaattaa attactgtca 61gcagctaacc aatggcagga gccgtttcat cggctgcaca agcagcaaga tcaaaagtga 121gccttttctg attgctgcat agtgtcaatt ggccaatctc ttctcccagg gaaaaaaaaa 181agtaaatcaa acctttgaga agcatttgct ggttgaagtg ctttctgtct agtgaggggg 241tctgtggatt tctagtttat gataaatagg actttaaaaa ccagggacgg gagggcgagt 301gttcaggttc tagagctatg cagctggagc actgcctttc tccttctatc atgctctcca 361agaaatttct caatgtgagc agcagctacc cacattcagg cggatccgag cttgtcttgc 421acgatcatcc cattatctcg accactgaca acctggagag aagttcacct ttgaaaaaaa 481ttaccagggg gatgacgaat cagtcagata cagacaattt tcctgactcc aaggactcac 541caggggacgt ccagagaagt aaactctctc ctgtcttgga cggggtctct gagcttcgtc 601acagtttcga tggctctgct gcagatcgct acctcctctc tcagtccagc cagccacagt 661ctgcggccac tgctcccagt gccatgttcc cgtaccccgg ccagcacgga ccggcgcacc 721ccgccttctc catcggcagc cctagccgct acatggccca ccacccggtc atcaccaacg 781gagcctacaa cagcctcctg tccaactcct cgccgcaggg ataccccacg gccggctacc 841cctacccaca gcagtacggc cactcctacc aaggagctcc gttctaccag ttctcctcca 901cccagccggg gctggtgccc ggcaaagcac aggtgtacct gtgcaacagg cccctttggc 961tgaaatttca ccggcaccaa acggagatga tcatcaccaa acagggaagg cgcatgtttc 1021cttttttaag ttttaacatt tctggtctcg atcccacggc tcattacaat atttttgtgg 1081atgtgatttt ggcggatccc aatcactgga ggtttcaagg aggcaaatgg gttccttgcg 1141gcaaagcgga caccaatgtg caaggaaatc gggtctatat gcatccggat tcccccaaca 1201ctggggctca ctggatgcgc caagaaatct cttttggaaa attaaaactt acgaacaaca 1261aaggagcttc aaataacaat gggcagatgg tggttttaca gtccttgcac aagtaccagc 1321cccgcctgca tgtggtggaa gtgaacgagg acggcacgga ggacactagc cagcccggcc 1381gcgtgcagac gttcactttc cctgagactc agttcatcgc cgtcaccgcc taccagaaca 1441cggatattac acaactgaaa atagatcaca acccttttgc aaaaggattt cgggataatt 1501atgacacgat ctacaccggc tgtgacatgg accgcctgac cccctcgccc aacgactcgc 1561cgcgctcgca gatcgtgccc ggggcccgct acgccatggc cggctctttc ctgcaggacc 1621agttcgtgag caactacgcc aaggcccgct tccacccggg cgcgggcgcg ggccccgggc 1681cgggtacgga ccgcagcgtg ccgcacacca acgggctgct gtcgccgcag caggccgagg 1741acccgggcgc gccctcgccg caacgctggt ttgtgacgcc ggccaacaac cggctggact 1801tcgcggcctc ggcctatgac acggccacgg acttcgcggg caacgcggcc acgctgctct 1861cttacgcggc ggcgggcgtg aaggcgctgc cgctgcaggc tgcaggctgc actggccgcc 1921cgctcggcta ctacgccgac ccgtcgggct ggggcgcccg cagtcccccg cagtactgcg 1981gcaccaagtc gggctcggtg ctgccctgct ggcccaacag cgccgcggcc gccgcgcgca 2041tggccggcgc caatccctac ctgggcgagg aggccgaggg cctggccgcc gagcgctcgc 2101cgctgccgcc cggcgccgcc gaggacgcca agcccaagga cctgtccgat tccagctgga 2161tcgagacgcc ctcctcgatc aagtccatcg actccagcga ctcggggatt tacgagcagg 2221ccaagcggag gcggatctcg ccggccgaca cgcccgtgtc cgagagttcg tccccgctca 2281agagcgaggt gctggcccag cgggactgcg agaagaactg cgccaaggac attagcggct 2341actatggctt ctactcgcac agctaggccg cccctgcccg cccggccccg ccgcggcccg 2401gacccccagc cagcccctca cagctcttcc ccagctccgc ctccccacac tcctccttgc 2461gcacccactc attttatttg accctcgatg gccgtctgca gcgaataagt gcaggtctcc 2521gagcgtgatt ttaacctttt ttgcacagca gtctctgcaa ttagctcacc gaccttcaac 2581tttgctgtaa accttttggt tttcctactt actcttcttc tgtggagtta tcctcctaca 2641attcccctcc ccctcgtctt tctcttacct cctacttctc tttcttgtaa tgaaactctt 2701cacctttagg agacctgggc agtcctgtca ggcagcagcg attccgaccc gccaagtctc 2761ggcctccaca ttaaccatag gatgttgact ctagaacctg gacccaccca gcgcgtcctt 2821tcttatcccc gagtggatgg atggatggat ggatggtagg gatgttaata attttagtgg 2881aacaaagcct gtgaaatgat tgtacatagt gttaatttat tgtaacgaat ggctagtttt 2941tattctcgtc aaggcacaaa accagttcat gcttaacctt tttttccttt cctttctttg 3001cttttctttc tctcctctca tactttctct tctctctctt ttaattttct tgtgagataa 3061tattctaaga ggctctagaa acatgaaata ctcagtagtg atgggtttcc cacttctcct 3121caatccgttg catgaaataa ttactatgtg ccctaatgca cacaaatagc taaggagaat 3181ccacccaaac acctttaaag gataggtgtc tgttcatagg caagtcgatt aagtggcatg 3241atgcctgcaa agcaaagtca actggagttg tatgttcccc ccaccttcta aatagaatag 3301ctcgacatca gcaatattat tttgccttat ttgtttttcc ccaaagtgcc aaatccatta 3361ctggtctgtg caggtgccaa atatgctgac aaactgtttc tgaatatctt tcagtacccc 3421ttcaccttta tatgctgtaa atctttgtaa tgaatactct attaatgata tagatgactg 3481aattgttggt aactatagtg tagtctagtg aagatgaatt gtgtgagttg tatattttac 3541tgcattttag ttttgaaaat gacttcccca ccacctagaa acagctgaaa tttgacttcc 3601ttgggagaac actagcatta atgcaagtaa gactgatttt cccctaagtc ttgttatatt 3661tgataaggag cattaatccc cctggaaata gattagtagg atttctaatg ttgtgtagca 3721aacctatact tttttgtatt taaaaattaa tgtgaaatat gcatcataca caatattcaa 3781tctagattcc agtccatggg gggatttttc ctaataggaa ttcagggtct aaacgtgtgt 3841atattttggc tcttctgtaa atctaatgtt gtgattttta tatttgtttc gttttgtctg 3901tgaactgaat aatttataca agaacacact ccattgagaa acgttttgtt ttttgctcgt 3961ttgtatcgtc tgtgtataac aagtaaaata aacctggtaa aaacgc

By “SLC1A3 polypeptide” (or solute carrier family 1; glial high affinityglutamate transporter member 3 (SLC1A3)) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. BAG35230.1.

MTKSNGEEPKMGGRMERFQQGVRKRTLLAKKKVQNITKEDVKSYLFRNAFVLLTVTAVIVGTILGFTLRPYRMSYREVKYFSFPGELLMRMLQMLVLPLIISSLVTGMAALDSKASGKMGMRAVVYYMTTTIIAVVIGIIIVIIIHPGKGTKENMHREGKIVRVTAADAFLDLIRNMFPPNLVEACFKQFKTNYEKRSFKVPIQANETLVGAVINNVSEAMETLTRITEELVPVPGSVNGVNALGLVVFSMCFGFVIGNMKEQGQALREFFDSLNEAIMRLVAVIMWYAPVGILFLIAGKIVEMEDMGVIGGQLAMYTVTVIVGLLIHAVIVLPLLYFLVTRKNPWVFIGGLLQALITALGTSSSSATLPITFKCLEENNGVDKRVTRFVLPVGATINMDGTALYEALAAIFIAQVNNFELNFGQIITISITATAASIGAAGIPQAGLVTMVIVLTSVGLPTDDITLIIAVDWFLDRLRTTTNVLGDSLGAGIVEHLSRHELKNRDVEMGNSVIEENEMKKPYQLIAQDNETEKPIDSETKM

By “SLC1A3 polynucleotide” (or solute carrier family 1; glial highaffinity glutamate transporter member 3 (SLC1A3)) is meant apolynucleotide encoding an SLC1A3 polypeptide. An exemplary SLC1A3nucleic acid molecule (e.g., mRNA) is provided at NCBI Accession No.AK312304.

1 gatagtaact tgcagtttca gagcacatgc acactgtcag ggctagcctg cctgcttacg 61cgcgctgcgg attgttgctc cgttgtacct gctggggaat tcacctcgtt actgcttgat 121atcttccacc ccttacaaaa tcagaaaagt tgtgttttct aataccaaag aggaggtttg 181gctttctgtg ggtgattccc agacactgaa gtgcaaagaa gagaccctcc tagaaaagta 241aaatatgact aaaagcaatg gagaagagcc caagatgggg ggcaggatgg agagattcca 301gcagggagtc cgtaaacgca cacttttggc caagaagaaa gtgcagaaca ttacaaagga 361ggatgttaaa agttacctgt ttcggaatgc ttttgtgctg ctcacagtca ccgctgtcat 421tgtgggtaca atccttggat ttaccctccg accatacaga atgagctacc gggaagtcaa 481gtacttctcc tttcctgggg aacttctgat gaggatgtta cagatgctgg tcttaccact 541tatcatctcc agtcttgtca caggaatggc ggcgctagat agtaaggcat cagggaagat 601gggaatgcga gctgtagtct attatatgac taccaccatc attgctgtgg tgattggcat 661aatcattgtc atcatcatcc atcctgggaa gggcacaaag gaaaacatgc acagagaagg 721caaaattgta cgagtgacag ctgcagatgc cttcctggac ttgatcagga acatgttccc 781tccaaatctg gtagaagcct gctttaaaca gtttaaaacc aactatgaga agagaagctt 841taaagtgccc atccaggcca acgaaacgct tgtgggtgct gtgataaaca atgtgtctga 901ggccatggag actcttaccc gaatcacaga ggagctggtc ccagttccag gatctgtgaa 961tggagtcaat gccctgggtc tagttgtctt ctccatgtgc ttcggttttg tgattggaaa 1021catgaaggaa caggggcagg ccctgagaga gttctttgat tctcttaacg aagccatcat 1081gagactggta gcagtaataa tgtggtatgc ccccgtgggt attctcttcc tgattgctgg 1141gaagattgtg gagatggaag acatgggtgt gattgggggg cagcttgcca tgtacaccgt 1201gactgtcatt gttggcttac tcattcacgc agtcatcgtc ttgccactcc tctacttctt 1261ggtaacacgg aaaaaccctt gggtttttat tggagggttg ctgcaagcac tcatcaccgc 1321tctggggacc tcttcaagtt ctgccaccct acccatcacc ttcaagtgcc tggaagagaa 1381caatggcgtg gacaagcgcg tcaccagatt cgtgctcccc gtaggagcca ccattaacat 1441ggatgggact gccctctatg aggctttggc tgccattttc attgctcaag ttaacaactt 1501tgaactgaac ttcggacaaa ttattacaat cagcatcaca gccacagctg ccagtattgg 1561ggcagctgga attcctcagg cgggcctggt cactatggtc attgtgctga catctgtcgg 1621cctgcccact gacgacatca cgctcatcat cgcggtggac tggttcctgg atcgcctccg 1681gaccaccacc aacgtactgg gagactccct gggagctggg attgtggagc acttgtcacg 1741acatgaactg aagaacagag atgttgaaat gggtaactca gtgattgaag agaatgaaat 1801gaagaaacca tatcaactga ttgcacagga caatgaaact gagaaaccca tcgacagtga 1861aaccaagatg tag

By “TH polypeptide” (or tyrosine hydroxylase) is meant a polypeptide orfragment thereof having at least about 85% amino acid identity to NCBIAccession No. AAI43612.1.

MPTPDATTPQAKGFRRAVSELDAKQAEAIMSPRFIGRRQSLIEDARKEREAAVAAAAAAVPSEPGDPLEAVAFEEKEGKAVLNLLFSPRATKPSALSRAVKVFETFEAKIHHLETRPAQRPRAGGPHLEYFVRLEVRRGDLAALLSGVRQVSEDVRSPAGPKVPWFPRKVSELDKCHHLVTKFDPDLDLDHPGFSDQVYRQRRKLIAEIAFQYRHGDPIPRVEYTAEEIATWKEVYTTLKGLYATHACGEHLEAFALLERFSGYREDNIPQLEDVSRFLKERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLADRTFAQFSQDIGLASLGASDEEIEKLSTLYWFTVEFGLCKQNGEVKAYGAGLLSSYGELLHCLSEEPEIRAFDPEAAAVQPYQDQTYQSVYFVSESFSDAKDKLRSYASRIQRPFSVKFDPYTLAIDVLDSPQAVRRSLEGVQDELDTLAHALSAIG

By “TH polynucleotide” (or tyrosine hydroxylase) is meant apolynucleotide encoding an TH polypeptide. An exemplary TH nucleic acidmolecule (e.g., mRNA) is provided at NCBI Accession No. BC143611.

   1 acccagaggg ggctttgacg tcagctcagc ttataagagg ctgctgggcc agggctgtgg  61 agacggagcc cggacctcca cactgagcca tgcccacccc cgacgccacc acgccacagg 121 ccaagggctt ccgcagggcc gtgtctgagc tggacgccaa gcaggcagag gccatcatgt 181 ccccgcggtt cattgggcgc aggcagagcc tcatcgagga cgcccgcaag gagcgggagg 241 cggcggtggc agcagcggcc gctgcagtcc cctcggagcc cggggacccc ctggaggctg 301 tggcctttga ggagaaggag gggaaggccg tgctaaacct gctcttctcc ccgagggcca 361 ccaagccctc ggcgctgtcc cgagctgtga aggtgtttga gacgtttgaa gccaaaatcc 421 accatctaga gacccggccc gcccagaggc cgcgagctgg gggcccccac ctggagtact 481 tcgtgcgcct cgaggtgcgc cgaggggacc tggccgccct gctcagtggt gtgcgccagg 541 tgtcagagga cgtgcgcagc cccgcggggc ccaaggtccc ctggttccca agaaaagtgt 601 cagagctgga caagtgtcat cacctggtca ccaagttcga ccctgacctg gacttggacc 661 acccgggctt ctcggaccag gtgtaccgcc agcgcaggaa gctgattgct gagatcgcct 721 tccagtacag gcacggcgac ccgattcccc gtgtggagta caccgccgag gagattgcca 781 cctggaagga ggtctacacc acgctgaagg gcctctacgc cacgcacgcc tgcggggagc 841 acctggaggc ctttgctttg ctggagcgct tcagcggcta ccgggaagac aatatccccc 901 agctggagga cgtctcccgc ttcctgaagg agcgcacggg cttccagctg cggcctgtgg 961 ccggcctgct gtccgcccgg gacttcctgg ccagcctggc cttccgcgtg ttccagtgca1021 cccagtatat ccgccacgcg tcctcgccca tgcactcccc tgagccggac tgctgccacg1081 agctgctggg gcacgtgccc atgctggccg accgcacctt cgcgcagttc tcgcaggaca1141 ttggcctggc gtccctgggg gcctcggatg aggaaattga gaagctgtcc acgctgtact1201 ggttcacggt ggagttcggg ctgtgtaagc agaacgggga ggtgaaggcc tatggtgccg1261 ggctgctgtc ctcctacggg gagctcctgc actgcctgtc tgaggagcct gagattcggg1321 ccttcgaccc tgaggctgcg gccgtgcagc cctaccaaga ccagacgtac cagtcagtct1381 acttcgtgtc tgagagcttc agtgacgcca aggacaagct caggagctat gcctcacgca1441 tccagcgccc cttctccgtg aagttcgacc cgtacacgct ggccatcgac gtgctggaca1501 gcccccaggc cgtgcggcgc tccctggagg gtgtccagga tgagctggac acccttgccc1561 atgcgctgag tgccattggc taggtgcacg gcgtccctga gggcccttcc caacctcccc1621 tggtcctgc

By “Neurofilament 200 polypeptide” (or neurofilament heavy (NEFH)) ismeant a polypeptide or fragment thereof having at least about 85% aminoacid identity to NCBI Accession No. NP_066554.2.

MMSFGGADALLGAPFAPLHGGGSLHYALARKGGAGGTRSAAGSSSGFHSWTRTSVSSVSASPSRFRGAGAASSTDSLDTLSNGPEGCMVAVATS RSEKEQLQALNDRFAGYIDKVRQLEAHNRSLEGEAAALRQQQAGRSAMGELYEREVRE MRGAVLRLGAARGQLRLEQEHLLEDIAHVRQRLDDEARQREEAEAAARALARFAQEAE AARVDLQKKAQALQEECGYLRRHHQEEVGELLGQIQGSGAAQAQMQAETRDALKCDVT SALREIRAQLEGHAVQSTLQSEEWFRVRLDRLSEAAKVNTDAMRSAQEEITEYRRQLQ ARTTELEALKSTKDSLERQRSELEDRHQADIASYQEAIQQLDAELRNTKWEMAAQLRE YQDLLNVKMALDIEIAAYRKLLEGEECRIGFGPIPFSLPEGLPKIPSVSTHIKVKSEEKIKVVEKSEKETVIVEEQTEETQVTEEVTEEEEKEAKEEEGKEEEGGEEEEAEGGEEETKSPPAEEAASPEKEAKSPVKEEAKSPAEAKSPEKEEAKSPAEVKSPEKAKSPAKEEAKSPPEAKSPEKEEAKSPAEVKSPEKAKSPAKEEAKSPAEAKSPEKAKSPVKEEAKSPAEAKSPVKEEAKSPAEVKSPEKAKSPTKEEAKSPEKAKSPEKEEAKSPEKAKSPVKAEA KSPEKAKSPVKAEAKSPEKAKSPVKEEAKSPEKAKSPVKEEAKSPEKAKSPVKEEAKT PEKAKSPVKEEAKSPEKAKSPEKAKTLDVKSPEAKTPAKEEARSPADKFPEKAKSPVK EEVKSPEKAKSPLKEDAKAPEKEIPKKEEVKSPVKEEEKPQEVKVKEPPKKAEEEKAP ATPKTEEKKDSKKEEAPKKEAPKPKVEEKKEPAVEKPKESKVEAKKEEAEDKKKVPTP EKEAPAKVEVKEDAKPKEKTEVAKKEPDDAKAKEPSKPAEKKEAAPEKKDTKEEKAKK PEEKPKTEAKAKEDDKTLSKEPSKPKAEKAEKSSSTDQKDSKPPEKATEDKAAKGK

By “Neurofilament 200 polynucleotide” (or neurofilament heavy (NEFH)) ismeant a polynucleotide encoding an Neurofilament 200 polypeptide. Anexemplary Neurofilament 200 nucleic acid molecule (e.g., mRNA) isprovided at NCBI Accession No. NM_021076.

   1 aaaagggccg gcgccctggt gctgccgcag tgcctcccgc cccgtcccgg cctcgcgcac  61 ctgctcaggc catgatgagc ttcggcggcg cggacgcgct gctgggcgcc ccgttcgcgc 121 cgctgcatgg cggcggcagc ctccactacg cgctagcccg aaagggtggc gcaggcggga 181 cgcgctccgc cgctggctcc tccagcggct tccactcgtg gacacggacg tccgtgagct 241 ccgtgtccgc ctcgcccagc cgcttccgtg gcgcaggcgc cgcctcaagc accgactcgc 301 tggacacgct gagcaacggg ccggagggct gcatggtggc ggtggccacc tcacgcagtg 361 agaaggagca gctgcaggcg ctgaacgacc gcttcgccgg gtacatcgac aaggtgcggc 421 agctggaggc gcacaaccgc agcctggagg gcgaggctgc ggcgctgcgg cagcagcagg 481 cgggccgctc cgctatgggc gagctgtacg agcgcgaggt ccgcgagatg cgcggcgcgg 541 tgctgcgcct gggcgcggcg cgcggtcagc tacgcctgga gcaggagcac ctgctcgagg 601 acatcgcgca cgtgcgccag cgcctagacg acgaggcccg gcagcgagag gaggccgagg 661 cggcggcccg cgcgctggcg cgcttcgcgc aggaggccga ggcggcgcgc gtggacctgc 721 agaagaaggc gcaggcgctg caggaggagt gcggctacct gcggcgccac caccaggaag 781 aggtgggcga gctgctcggc cagatccagg gctccggcgc cgcgcaggcg cagatgcagg 841 ccgagacgcg cgacgccctg aagtgcgacg tgacgtcggc gctgcgcgag attcgcgcgc 901 agcttgaagg ccacgcggtg cagagcacgc tgcagtccga ggagtggttc cgagtgaggc 961 tggaccgact gtcggaggca gccaaggtga acacagacgc tatgcgctca gcgcaggagg1021 agataactga gtaccggcgt cagctgcagg ccaggaccac agagctggag gcactgaaaa1081 gcaccaagga ctcactggag aggcagcgct ctgagctgga ggaccgtcat caggccgaca1141 ttgcctccta ccaggaagcc attcagcagc tggacgctga gctgaggaac accaagtggg1201 agatggccgc ccagctgcga gaataccagg acctgctcaa tgtcaagatg gctctggata1261 tagagatagc cgcttacaga aaactcctgg aaggtgaaga gtgtcggatt ggctttggcc1321 caattccttt ctcgcttcca gaaggactcc ccaaaattcc ctctgtgtcc actcacataa1381 aggtgaaaag cgaagagaag atcaaagtgg tggagaagtc tgagaaagaa actgtgattg1441 tggaggaaca gacagaggag acccaagtga ctgaagaagt gactgaagaa gaggagaaag1501 aggccaaaga ggaggagggc aaggaggaag aagggggtga agaagaggag gcagaagggg1561 gagaagaaga aacaaagtct cccccagcag aagaggctgc atccccagag aaggaagcca1621 agtcaccagt aaaggaagag gcaaagtcac cggctgaggc caagtcccca gagaaggagg1681 aagcaaaatc cccagccgaa gtcaagtccc ctgagaaggc caagtctcca gcaaaggaag1741 aggcaaagtc accgcctgag gccaagtccc cagagaagga ggaagcaaaa tctccagctg1801 aggtcaagtc ccccgagaag gccaagtccc cagcaaagga agaggcaaag tcaccggctg1861 aggccaagtc tccagagaag gccaagtccc cagtgaagga agaagcaaag tcaccggctg1921 aggccaagtc cccagtgaag gaagaagcaa aatctccagc tgaggtcaag tccccggaaa1981 aggccaagtc tccaacgaag gaggaagcaa agtcccctga gaaggccaag tccccagaga2041 aggaagaggc caagtcccct gagaaggcca agtccccagt gaaggcagaa gcaaagtccc2101 ctgagaaggc caagtcccca gtgaaggcag aagcaaagtc ccctgagaag gccaagtccc2161 cagtgaagga agaagcaaag tcccctgaga aggccaagtc cccagtgaag gaagaagcaa2221 agtcccctga gaaggccaag tccccagtga aggaagaagc aaagaccccc gagaaggcca2281 agtccccagt gaaggaagaa gctaagtccc cagagaaggc caagtcccca gagaaggcca2341 agactcttga tgtgaagtct ccagaagcca agactccagc gaaggaggaa gcaaggtccc2401 ctgcagacaa attccctgaa aaggccaaaa gccctgtcaa ggaggaggtc aagtccccag2461 agaaggcgaa atctcccctg aaggaggatg ccaaggcccc tgagaaggag atcccaaaaa2521 aggaagaggt gaagtcccca gtgaaggagg aggagaagcc ccaggaggtg aaagtcaaag2581 agcccccaaa gaaggcagag gaagagaaag cccctgccac accaaaaaca gaggagaaga2641 aggacagcaa gaaagaggag gcacccaaga aggaggctcc aaagcccaag gtggaggaga2701 agaaggaacc tgctgtcgaa aagcccaaag aatccaaagt tgaagccaag aaggaagagg2761 ctgaagataa gaaaaaagtc cccaccccag agaaggaggc tcctgccaag gtggaggtga2821 aggaagacgc taaacccaaa gaaaagacag aggtagccaa gaaggaacca gatgatgcca2881 aggccaagga acccagcaaa ccagcagaga agaaggaggc agcaccggag aaaaaagaca2941 ccaaggagga gaaggccaag aagcctgagg agaaacccaa gacagaggcc aaagccaagg3001 aagatgacaa gaccctctca aaagagccta gcaagcctaa ggcagaaaag gctgaaaaat3061 cctccagcac agaccaaaaa gacagcaagc ctccagagaa ggccacagaa gacaaggccg3121 ccaaggggaa gtaaggcagg gagaaaggaa catccggaac agccaaagaa actcagaaga3181 gtcccggagc tcaaggatca gagtaacaca attttcactt tttctgtctt tatgtaagaa3241 gaaactgctt agatgacggg gcctccttct tcaaacagga atttctgtta gcaatatgtt3301 agcaagagag ggcactccca ggcccctgcc cccaggccct ccccaggcga tggacaatta3361 tgatagctta tgtagctgaa tgtgatacat gccgaatgcc acacgtaaac acttgactat3421 aaaaactgcc cccctccttt ccaaataagt gcatttattg cctctatgtg caactgacag3481 atgaccgcaa taatgaatga gcagttagaa atacattatg cttgagatgt cttaacctat3541 tcccaaatgc cttctgtttt ccaaaggagt ggtcaagccc ttgcccagag ctctctattc3601 tggaagagcg gtccaggtgg ggccggggac tggccactga attatgccag ggcgcacttt3661 ccactggagt tcactttcaa ttgcttctgt gcaataaaac caagtgctta taaaatgaaa3721 a

By “Map2” (or microtubule-associated protein 2) is meant a polypeptideor fragment thereof having at least about 85% amino acid identity toNCBI Accession No. AAH38857.1.

MADERKDEAKAPHWTSAPLTEASAHSHPPEIKDQGGAGEGLVRSANGFPYREDEEGAFGEHGSQGTYSNTKENGINGELTSADRETAEEVSARI VQVVTAEAVAVLKGEQEKEAQHKDQTAALPLAAEETANLPPSPPPSPASEQTVTVEEA AGGESALAPSVFKQAKDKVSNSTLSKIPALQGSTKSPRYSSACPSTTKRATFSDSLLIQPTSAGSTDRLPYSKSGNKDGVTKSPEKRSSLPRPSSILPPRRGVSGDRDENSFSLNSSISSSARRTTRSEPIRRAGKSGTSTPTTPGSTAITPGTPPSYSSRTPGTPGTPSYPRTPHTPGTPKSAILVPSEKKVAIIRTPPKSPATPKQLRLINQPLPDLKNVKSKIGSTDNIKYQPKGGQVRILNKKIDFSKVQSRCGSKDNIKHSAGGGNVQIVTKKIDLSHVTSKCGSLKNIRHRPGGGRVKIESVKLDFKEKAQAKVGSLDNAHHVPGGGNVKIDSQKLNFREHAKARVDHGAEIITQSPGRSSVASPRRLSNVSSSGSINLLESPQLATLAEDVTAALAKQG L

By “Map2 polynucleotide” (or microtubule-associated protein 2) is meanta polynucleotide encoding an Map2 polypeptide. An exemplary Map2 nucleicacid molecule (e.g., mRNA) is provided at NCBI Accession No. BC038857.

   1 ggcgctcggg ctgcgcgggc tctgggcagc agcagcagca gcagcagcat cctctcttcc  61 tttacttccc ttccgcttct ttctcttcct tctccttctt tttccccccc ctccccttct 121 tcccctaacc cttctacccc tctccttttt ctccggaggg cgctaagtcc gtgagcggtg 181 gcagtcgcga ccgcgggtgc atccagtttc tgcgcccaga ttttattgat ctaatccaaa 241 gtatcttata acttctggct ggaattaaga ttcttcagct tgtctctaac cgaggaagca 301 ttgattggga gctactcatt cagaaaatta aaagaaagaa gccagaaaat attatcaacc 361 ctttgagaac acgacacaac gaactttata ttttaccact tccttgaata gttgcaggag 421 aaataacaag gcattgaaga atggcagatg aacggaaaga cgaagcaaag gcacctcact 481 ggacctcagc accgctaaca gaggcatctg cacactcaca tccacctgag attaaggatc 541 aaggcggagc aggggaagga cttgtccgaa gcgccaatgg attcccatac agggaggatg 601 aagagggtgc ctttggagag catgggtcac agggcaccta ttcaaatacc aaagagaatg 661 ggatcaacgg agagctgacc tcagctgaca gagaaacagc agaggaggtg tctgcaagga 721 tagttcaagt agtcactgct gaggctgtag cagtcctgaa aggtgaacaa gagaaagaag 781 ctcaacataa agaccagact gcagctctgc ctttagcagc tgaagaaaca gctaatctgc 841 ctccttctcc acccccatca cctgcctcag aacagactgt cacagtggag gaagcagcag 901 gtggggaatc agctctggct cccagtgtat ttaaacaggc aaaggacaaa gtctctaatt 961 ctaccttgtc aaagattcct gctttacagg gtagcacaaa gtccccaaga tacagctcag1021 cctgccctag cacgactaaa agggctacat tttctgacag tttattaata cagcccacct1081 cagcaggctc cacagaccgt ttgccatact caaaatcagg gaacaaggac ggagtaacca1141 agagcccaga aaagcgctct tctctcccaa gaccttcctc cattctccct cctcggcgag1201 gtgtgtcagg agacagagat gagaattcct tctctctcaa cagttctatc tcttcttcag1261 cacggcggac caccaggtca gagccaattc gcagagcagg gaagagtggt acctcaacac1321 ccactacccc tgggtctact gccatcactc ctggcacccc accaagttat tcttcacgca1381 caccaggcac tcctggaacc cctagctatc ccaggacccc tcacacacca ggaaccccca1441 agtctgccat cttggtgccg agtgagaaga aggtcgccat catacgtact cctccaaaat1501 ctcctgcgac tcccaagcag cttcggctta ttaaccaacc actgccagac ctgaagaatg1561 tcaaatccaa aatcggatca acagacaaca tcaaatacca gcctaaaggg gggcaggtta1621 ggattttaaa caagaagatc gattttagca aagttcagtc cagatgtggt tccaaggata1681 acatcaaaca ttcggctggg ggcggaaatg tacaaattgt taccaagaaa atagacctaa1741 gccatgtgac atccaaatgt ggctctctga agaacatccg ccacaggcca ggtggcggac1801 gtgtgaaaat tgagagtgta aaactagatt tcaaagaaaa ggcccaagct aaagttggtt1861 ctcttgataa tgctcatcat gtacctggag gtggtaatgt caagattgac agccaaaagt1921 tgaacttcag agagcatgct aaagcccgtg tggaccatgg ggctgagatc attacacagt1981 ccccaggcag atccagcgtg gcatcacccc gacgactcag caatgtctcc tcgtctggaa2041 gcatcaacct gctcgaatct cctcagcttg ccactttggc tgaggatgtc actgctgcac2101 tcgctaagca gggcttgtga atatttctca tttagcattg aaataataat atttaggcat2161 gagctcttgg caggagtggg ctctgagcag ttgttatatt cattctttat aaaccataaa2221 ataaataatc tcatccccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa2281 aaaaaa

By “DCX” (or doublecortin) is meant a polypeptide or fragment thereofhaving at least about 85% amino acid identity to NCBI Accession No.NP_835366.1.

MELDFGHFDERDKTSRNMRGSRMNGLPSPTHSAHCSFYRTRTLQALSNEKKAKKVRFYRNGDRYFKGIVYAVSSDRFRSFDALLADLTRSLSDN INLPQGVRYIYTIDGSRKIGSMDELEEGESYVCSSDNFFKKVEYTKNVNPNWSVNVKT SANMKAPQSLASSNSAQARENKDFVRPKLVTIIRSGVKPRKAVRVLLNKKTAHSFEQV LTDITEAIKLETGVVKKLYTLDGKQVTCLHDFFGDDDVFIACGPEKFRYAQDDFSLDE NECRVMKGNPSATAGPKASPTPQKTSAKSPGPMRRSKSPADSANGTSSSQLSTPKSKQ SPISTPTSPGSLRKHKDLYLPLSLDDSDSLGDSM

By “DCX polynucleotide” (or doublecortin) is meant a polynucleotideencoding an DCX polypeptide. An exemplary DCX nucleic acid molecule(e.g., mRNA) is provided at NCBI Accession No. NM 178153.

   1 ctggcaggaa tttcttgctt ggagctcaga caacaaaggc atagagagat tggttttctt  61 tctctcagca tctccaccca accagcagaa aaccggtctc tgaggttcca ccaaaatatg 121 gaacttgatt ttggacactt tgacgaaaga gataagacat ccaggaacat gcgaggctcc 181 cggatgaatg ggttgcctag ccccactcac agcgcccact gtagcttcta ccgaaccaga 241 accttgcagg cactgagtaa tgagaagaaa gccaagaagg tacgtttcta ccgcaatggg 301 gaccgctact tcaaggggat tgtgtacgct gtgtcctctg accgttttcg cagctttgac 361 gccttgctgg ctgacctgac gcgatctctg tctgacaaca tcaacctgcc tcagggagtg 421 cgttacattt acaccattga tggatccagg aagatcggaa gcatggatga actggaggaa 481 ggggaaagct atgtctgttc ctcagacaac ttctttaaaa aggtggagta caccaagaat 541 gtcaatccca actggtctgt caacgtaaaa acatctgcca atatgaaagc cccccagtcc 601 ttggctagca gcaacagtgc acaggccagg gagaacaagg actttgtgcg ccccaagctg 661 gttaccatca tccgcagtgg ggtgaagcct cggaaggctg tgcgtgtgct tctgaacaag 721 aagacagccc actcttttga gcaagtcctc actgatatca cagaagccat caaactggag 781 accggggttg tcaaaaaact ctacactctg gatggaaaac aggtaacttg tctccatgat 841 ttctttggtg atgatgatgt gtttattgcc tgtggtcctg aaaaatttcg ctatgctcag 901 gatgattttt ctctggatga aaatgaatgc cgagtcatga agggaaaccc atcagccaca 961 gctggcccaa aggcatcccc aacacctcag aagacttcag ccaagagccc tggtcctatg1021 cgccgaagca agtctccagc tgactcagca aacggaacct ccagcagcca gctctctacc1081 cccaagtcta agcagtctcc catctctacg cccaccagtc ctggcagcct ccggaagcac1141 aaggacctgt acctgcctct gtccttggat gactcggact cgcttggtga ttccatgtaa1201 aggaggggag agtgctcaga gtccagagta caaatccaag cctatcattg tagtagggta1261 cttctgctca agtgtccaac agggctattg gtgctttcaa gtttttattt tgttgttgtt1321 gttattttga aaaacacatt gtaatatgtt gggtttattt tcctgtgatt tctcctctgg1381 gccactgatc cacagttacc aattatgaga gatagattga taaccatcct ttggggcagc1441 attccaggga tgcaaaatgt gctagtccat gacctttcaa tggaaagctt aggtgcctgc1501 gttatatttg ccctgtctaa ttttgcccat acagtcttcc ttctgtagag ggctgtttac1561 atatacagca cttaaaatgt ttgtgtggga aaaaaaaaac tcattggcag atccaagaat1621 gacaaacaca agtgcccctt ttctctggat ctcaagaatg gtggaggacc ctggaaggac1681 agcaaggcag ctccccagcc tcactcttca ctcctgattg aggcccgggt ttgttgtcca1741 gcaccaattc tggctgtcaa tggggagaaa taaaccaaca acttataatt gtgacaccag1801 atgcttagga tcctggtgct gggttagcta agagaataga cagaattgga aaatactgca1861 gacatttccg aagagtttat aaagcacagt gaattcctgg tcaatctctc cactgaggca1921 atttggaatc aataagcaat tgataatagt ttggagtaag ggacttcata tacctgattc1981 ctctagaagg ctgtctaaca taccacatga ttacatgaac tgtatggtat ccatctatct2041 ctgttctatt gaatgccttg ttaacagcca acactgaaaa cactgtgaga atttgttttc2101 aggtctgaca cctttcagtc tctttttata gcaagaaatc aatatccttt ttataaaaat2161 tcatgtctgt atttcaggag caaactcttc aggctccttt tttataaact ggtgattttt2221 cttttgtcta aaaaacacat gaagaaaatt taccaaaaaa aaaaaaaaaa gcagaagaat2281 aatgtagttt agaaattatg ctgtcactgc caaacagtaa cctccaggag aaaacaagat2341 gaatagcaga ggccaattca atagaatcag ttttttgata gctttttaac agttatgctt2401 gcattaataa tttcaatgtg gaccagacat tctaattata ttttaaatga aatgttacag2461 catattttaa gcaactcttt ttatctataa tcctaatatt tcatactgaa gacacagaaa2521 tctttcactt gtctttaaca ttagaaagga tttctcttta ctaaggactg atcatttgaa2581 atagttttca gtcttttgag atacaggttt ataacactgc tttttttttt ctgtaatcat2641 agcccataat ggcaaagaca actaaattta agtgaaggtc atgcatgcca attctgtgtt2701 tgcttttagc agatatgaag atttccttat ttctttgtaa ttgtgcagat attttgaaag2761 gcacagcatt cgaagccaag ctgctgtttg gctactgaat ggcttgcagt tgttcctcca2821 ctctaaatgg aatgagcttg ctgtgtgtgt gtgtggtggt ggtgggaggg ggtggtgcat2881 gtgtgtgtgt gtgtgtgcat ctgcagctgc ttcaaaatta ggaaatacta caggacaccc2941 ctgtaatgga ttggtggcaa ctgggtggca ctgctgatgt gcactgtgta ggggggaacc3001 cagtggtggt ggggtagctc agatgcccct agacaagctt cagatgtctg tagctaccag3061 aaacattttc ggttcaggaa aagtgagatg atggtagtac tggtttctgg tgaaattgaa3121 gaaccccaaa tgatgaggat ctctttttgc cccctctcct ttttttgtag acccattcaa3181 aaccattaat aagcccattt tactaagccc ctatttcttt ctagaagctc agggttttct3241 tagtgcctcc cagaacattt tgtagttaat tgggaaaaag tgatacttgg attagggggt3301 gtgggcataa agaatggtgg gaggcctgat tttaaacttc aggccagaac ccccaatgac3361 tccacccata gtctcacttt aggtctcatt tagtccatca cctttatttt aagttgagga3421 agtggaggct ggtaaagagc aggaccagag gaagaatcca gatttcctta tgcttgggcc3481 tcacactagc tctctgagta tttccttgat tgcggtatat gtactactag aaaataccaa3541 atggatatat tttctttagg ataacctttg aaccaacaat cttcaataac aatagtacat3601 cttccatctt acttttaatc gagtataagg aaatgtttct ttatggccat tttggaggga3661 gcaggggatg aggcttggca tagtccaaaa tttaagtctc caataattaa ttgcatttta3721 aattggccca ctttcaaggc aatttttttt gtgtgtctgt aactgagctc ctccacccct3781 gtcattcact tccaatttta cccaatccaa ttttagcact caagttccat tgtgttaatt3841 tctgcacggt caacaaacat caagtcagca agcatttgcc accactccct atacttctcc3901 ctccttctta cacacacaca cacacacaca cacacaatcc atctcttgct tgttcctacc3961 tcctgatttt tcttccctac agaaatagaa atagggacaa agaaggggaa aatgtatata4021 ttggggctgg gctgaacaac taacttcata agtagtatta actaggggta aattgagaga4081 aaagctcctt ttctcttcac tgttttggaa aggatagcca ttagcatgac tgctttgtgt4141 ccttatggac tttagtatta gcctagattg aattatagcg ttttctagct ggaaggaacc4201 ttaagatcac atcatctact cctctactcc aaatttctca ttcttcaggc caggaaaccg4261 agacacagag gtaaagtaat ttccccaagg tcacacagct ggctggggca ggattgggtt4321 tacaacccac atctcctggc tcttattcca gggccttttc ccactaagta gtattgcctt4381 ccattaggct cctgagagtt atttctcagg gtcatgttgc atcttggagc cacatgctgc4441 tgccctgatc tcagtgggaa atccacccag caacctaata cagccccttt tccctgcatt4501 cacctggttc ccatccacat gggttgcaga tgtccttgaa gagagtgagg cattgagggc4561 caataggagc aatggggtcc ctggccttgt ccatctgatt caggagatca ctgctccatc4621 gtgaggagcc ctctgaatag ccccccactg aatgcttgcc ttgcccaaat ggaatggagg4681 aagattgatt ttctccatca gttcaccttg tgtcatctca taatggttgg tctttccagg4741 ctgagggaaa tgtttcttgt ttccagagta gaaaaagaaa gagtggaaca atagctttgt4801 tcatcctaac tttctgagat ggcttttcaa cattttaaaa aaactagtgt ggctaccatt4861 cactggcaat gatttctttt agaatatggg agtaagatga gctagagaaa ataacctggt4921 ctcactgtgg ttgccctcat ccacaatgtc cccaaagcca tcctgctctg atgaggacaa4981 tttccaggta taagcaaggg gctttgtgac aaaaatgtac cctggctgat gttaaacatt5041 ggctcctgtg tttgcaccaa aatagcaagc tgtgtgctct atacactctt cccatcgtct5101 tgtgtacact gctcctgtgg ccttccacag cagaaaccag ggcaaaaggg tccaaacaca5161 tggttttcct tgctgcaagg ctcttcctgg gaactaaggg ggtatttatt agttcagttc5221 taagagacct ccttctgggc ttaccccact cctcaggtac ttctctctcc ttcctccttc5281 tcctccacag tcacaagtaa ccaaggaacc tgaaagtgga tgtgtagcta tttgaagaag5341 gcaaggaacc ctgagattct tctttgaatc ctctagtcca agtcttagac cagtgattgg5401 tgcttacctt gaacaaaatt ttgtctgtgt tcctaatccc ttcaatactc tgggtacaat5461 gctcccaatc accctgcaca tttgattcta aatggctttt attttttaaa aatccatatc5521 cctaggacaa gagaacagga tgcctatatc cccaaaatga gctccaggac actgatggga5581 atgatcccaa agatcacccc acctcagaaa cgtctgtgcc aagagacttc cccagataga5641 aacactggga cagtggtttg aacgacttct tttatggttg tccagtttgc tatggaaata5701 aaaggcattg attttttaaa aagatgattg gaacctgtct ttggccacat agggccactt5761 ggatccattt ccaggcctta ctcatatatt gccttcactg aagggctttg gctttaagtc5821 ccagactggt ctcccaagtg aaccataagt gttttggagc tcatctgggg tgaggcatga5881 gaatgttgcc ccatctatcc cttcaggaaa aggtgccttc cctccctttc tcctaaagcc5941 tggtccccag aaattgtttt tgtctccaaa agtctagtat ggtctttata cacccagact6001 cttagtgttg cgtcctgcct tgtttccttg ttaaggatct atgcagacct cccgctttgg6061 cttagctagc gtgacattgg ctatcatttg acaagactaa cttttttttt tttttttttt6121 tgactgagtc tccctctgtc acctaggctg gagtgcagtg gcacaatctt ggctcgctgc6181 aaccttcacc cttcacctcc caggtcgaag cgattctcct gcctcagtct cccgagtagc6241 tgggattaca ggcgtgcgcc accaaatctg gctatttttt tattattatt atttttagta6301 gagatggggt ttcaccatgt tggccagact ggtcttgaac tcttggcctc aaattatctg6361 cccacctcgg cctcccaaag tgctgggatt acaggcatga gccaccatgc ccagctgaca6421 agactaattt tttatccctt ggtttattgg cttcaacatc ttctggaatc agaggtgatt6481 ttttcttacc ttggatgcct gagactaggg gagtatagaa ttccaattgg taattaaggc6541 atctttctgc tcctgatcag aagggcaggt tagttgggag aggtcagatg gcacaacaga6601 agtcaccttg taagtaaggc aaagacttga aggcattagc gtttctcatt actaggtcaa6661 taacctgagg gaatcaatgg ctttttgccg ctctacctct tgtgtatctc tttgactttt6721 ctttctctgt ctagtttcct ctgttctcag tttatattct atgttatcag tctctctttc6781 cacagtacaa acatccatcc tttctcctgt gcaattctgt ctctccctct tattatcttt6841 atttgtactt tttccttcct ccctgtctag gcattgggca tgtgcctctt cttagcctgt6901 gattttgcct tgggactgat gataaattat ttccagattc aatcagccct ggtcctaccc6961 cagtccaatc agaagtatgt tggtgggaat caacctgatc ctggcccttt cttcttctcc7021 attttcattc gtaatccccc tcagcagatc tttacaagca gtttccttat agctcatgta7081 tctttaggtc tttgccttcc aagcactgta cagaatactt tgtggttcct ttttagtctg7141 acattttgtg gagcagtgaa gcgtgctcag agacataatc agctgaagag aaaaaatcca7201 cccatggatt tatatcagct aaatactaat aattgatttt gtttgatgtg cccataattt7261 ttaaagctgc aatataatat aatgagggac cacaggtaat ttctcctgtc atttgttttg7321 gctggatggg ggtgggggag taattgctta aagttttacc attacacatt aaactctcta7381 taataatctt gtttggggct tgctaactgt tgagctgttt taactaaact ggtaggcaat7441 cggagttgat ttaaatgaaa agataattta acaaatctat actataaaaa gagacatttg7501 cttaattgac atgtattttt tccttctgag tcacctaaac atttactctt gacaccaact7561 gttcatgata ctgaatagac agtccatata agagaaatta gtggacctaa agaagccaga7621 ttgtaggtgt taatttatta aacagagtgc aaagcccttg gaaatgtcac tgcttggcaa7681 taccatatgg aatgccaaaa tttacaatga cttttcttta taagttatcc aaaagggatt7741 tgaacaagta agaggttatg ccaaaatgtc tccaatgtat ggtcctgtaa tatattgcag7801 cttgaagcca atgatccctt atgacttgta tacaactaat gcatgtttta ttgaattttg7861 catttcccac gtgtggtaag ttctttaaaa tgtttttgat cacctttttg tgccattaaa7921 cttgtacaga aaatgttttt atggccattt tcaaagggag aaagtttaaa atggaaacag7981 cccacccttt ctgccctata gctgtagtta gaattgagta cctgtagcaa aacagctgta8041 attggtggtt gtagtgttag aggtgttagc ttgctagtga ctagctttgg agagtaaatg8101 catggtattg tacatcacat ttcttaactc gttttaacct ctgaaaagaa tatattcttc8161 tttgtagtcc ttcttcccac ccccttgccc tctccctctc cctgctccca gttgtcttac8221 agttgtaaat atctgatttg aggcccaata actcttgcca agtaaagtca gcaaacaaca8281 aacaaaccaa aatgtgggga aaaggcattt ctcaaccatc tctcagcagt tattgatcat8341 ttcttaagga acagcattgt gatcaaagac tcaactttac gtaaaaatca gtggtaaatt8401 ggggttgtat ttggccattt gattacattt caggattgaa tagttttcag aatcacatgt8461 aatccaaaga cagtaggtag tgatgtccct tatccctgca gctgttttaa gatagagacc8521 tcagaagact ctgcttgacc gatgaccaat aattatttga aaaaaaaaga aaaaatgaga8581 gaaataaaac agatatttaa gaactttagc cacctattta gaatagttat agccagaaaa8641 aaaaacaagg gcatgagttc aaatgcatta ctatcagtgt cctaggcaat acctaaccta8701 ctctgaaatt gtgattcaaa agcagtattt caagaggcat tctccttttt tggtttgctg8761 accccacttg gactggtagg tttggtgagg cccccataaa ccagctggag cagacccttt8821 tcatctcctg tgcctgtaac acccctcttc ccccaccccc tccgcaattc aatgagggct8881 ttcttgggtc agaggacttc aaggttgtct agagaagttt gccatgtgtg taaggtgctg8941 tgaactgtga gtgctgaaga ttcgcagcat tcaataccag gcagccaaag agctgctctt9001 gcaattattt tggctctcaa gctctgttct tcatcgcatt ctcatttctg tgtacatttg9061 caagatgtgt gtaatgtcat tttccaaaaa taaaatttga tttcaataaa aaaaaaaaaa9121 aaaaaaaaaa aaaaa

By “GABRA1” (or gamma-aminobutyric acid (GABA) A receptor) is meant apolypeptide or fragment thereof having at least about 85% amino acididentity to NCBI Accession No. AAH30696.1.

MRKSPGLSDCLWAWILLLSTLTGRSYGQPSLQDELKDNTTVFTRILDRLLDGYDNRLRPGLGERVTEVKTDIFVTSFGPVSDHDMEYTIDVFFR QSWKDERLKFKGPMTVLRLNNLMASKIWTPDTFFHNGKKSVAHNMTMPNKLLRITEDG TLLYTMRLTVRAECPMHLEDFPMDAHACPLKFGSYAYTRAEVVYEWTREPARSVVVAE DGSRLNQYDLLGQTVDSGIVQSSTGEYVVMTTHFHLKRKIGYFVIQTYLPCIMTVILS QVSFWLNRESVPARTVFGVTTVLTMTTLSISARNSLPKVAYATAMDWFIAVCYAFVFS ALIEFATVNYFTKRGYAWDGKSVVPEKPKKVKDPLIKKNNTYAPTATSYTPNLARGDP GLATIAKSATIEPKEVKPETKPPEPKKTFNSVSKIDRLSRIAFPLLFGIFNLVYWATY LNREPQLK APTPHQ

By “GABRA1 polynucleotide” (or gamma-aminobutyric acid (GABA) Areceptor) is meant a polynucleotide encoding an GABRA1 polypeptide. Anexemplary GABRA1 nucleic acid molecule (e.g., mRNA) is provided at NCBIAccession No. BC030696.

   1 agcggagcgg gcgagcaagg gagcgagcag gacaggagcc tgatcccaca gctgctgctc  61 cagcccgcga tgaggaaaag tccaggtctg tctgactgtc tttgggcctg gatcctcctt 121 ctgagcacac tgactggaag aagctatgga cagccgtcat tacaagatga acttaaagac 181 aataccactg tcttcaccag gattttggac agactcctag atggttatga caatcgcctg 241 agaccaggat tgggagagcg tgtaaccgaa gtgaagactg atatcttcgt caccagtttc 301 ggacccgttt cagaccatga tatggaatat acaatagatg tatttttccg tcaaagctgg 361 aaggatgaaa ggttaaaatt taaaggacct atgacagtcc tccggttaaa taacctaatg 421 gcaagtaaaa tctggactcc ggacacattt ttccacaatg gaaagaagtc agtggcccac 481 aacatgacca tgcccaacaa actcctgcgg atcacagagg atggcacctt gctgtacacc 541 atgaggctga cagtgagagc tgaatgtccg atgcatttgg aggacttccc tatggatgcc 601 catgcttgcc cactaaaatt tggaagttat gcttatacaa gagcagaagt tgtttatgaa 661 tggaccagag agccagcacg ctcagtggtt gtagcagaag atggatcacg tctaaaccag 721 tatgaccttc ttggacaaac agtagactct ggaattgtcc agtcaagtac aggagaatat 781 gttgttatga ccactcattt ccacttgaag agaaagattg gctactttgt tattcaaaca 841 tacctgccat gcataatgac agtgattctc tcacaagtct ccttctggct caacagagag 901 tctgtaccag caagaactgt ctttggagta acaactgtgc tcaccatgac aacattgagc 961 atcagtgcca gaaactccct ccctaaggtg gcttatgcaa cagctatgga ttggtttatt1021 gccgtgtgct atgcctttgt gttctcagct ctgattgagt ttgccacagt aaactatttc1081 actaagagag gttatgcatg ggatggcaaa agtgtggttc cagaaaagcc aaagaaagta1141 aaggatcctc ttattaagaa aaacaacact tacgctccaa cagcaaccag ctacacccct1201 aatttggcca ggggcgaccc gggcttagcc accattgcta aaagtgcaac catagaacct1261 aaagaggtca agcccgaaac aaaaccacca gaacccaaga aaacctttaa cagtgtcagc1321 aaaattgacc gactgtcaag aatagccttc ccgctgctat ttggaatctt taacttagtc1381 tactgggcta cgtatttaaa cagagagcct cagctaaaag cccccacacc acatcaatag1441 atcttttact cacattctgt tgttcagtcc tctgcactgg gaatttattt atgttctcaa1501 cgcagtaatt cccatctgct ttattgcctc tgtcttaaag aatttgaaag tttccttatt1561 ttcataattc atttaagaac aagagacccc tgtctggcag tctggagcaa agcagactat1621 gcagcttgga gacaggattc tgacagagca agcgaaagag caaagtcatg tcagaaggag1681 acagaatgag agagaaaaga gggggaagat ggttcaaaga tacaagaaaa agtagaaaaa1741 aaaataacac ttaactaaaa cccctaggtc atttgtagat atatatttcc aaatattcta1801 aaaaagatac tgtatatgtc aaaaatattt ttatgtgaag gtgtttcaaa gggtaaatta1861 taaatgtttc atgaagaaaa aattttaaaa atctacgtct ttattacaca aactatggtg1921 tgcttatgtt tttgttttgc tttttaaact gatgtatagc tttaacattt tgtttccaaa1981 gctgaagatc cccattcttt ctctttgaaa aaaaaaaagg cctaatgcat tattttgtca2041 taaaatgcta ttttaaaatt catggaactt tcatacgtaa aggtgcagtt gctcattgta2101 gagcacattt agtccaatga agataaatgc tttaaatagt ttacttcact ttcatctgag2161 cttttaccac tagactcaag gaagaataat tttaacagac atgtatactc catagaaact2221 aaattaaaat agtttaaaaa tattcccttt ttcaccctat tttcagatag cacatgagcc2281 caacactcac ttaattctca ttatgaagat gtttttagag gggcaaaaat attttgcaag2341 ctctggaatt gttgaatgta ttcttttata taactacatt aaaagcttta gattgaaatt2401 tatgactagc aaacaaaaat agaatatata aacgatatat gtaaatatac agcatgagat2461 tgtacatttt ttactttttt aaaattgtgt tcttaaaata ttgtgtaaga atcactgcac2521 ttagctgttg gaatgttgtt aaatgctatg gaaatacatt tagaacctgc atttaagaac2581 agaacagcaa gtatgaacca catggaactt aaaacatatg ggtgtgaagt ccacttatgt2641 agacaaaact tataatttcc aaactgttgt ctagtataca gtgatcagtt gctctctgtt2701 caagtcattc cacacatttc cctattttag gctattataa tatagaaaga aaatgggaag2761 cattagttgg agctagaaaa tgaactgtat attattgcta tatttgctaa taccaactat2821 ttcaataagt gttgtaccat atgtagcatt aaatataaaa tacataaaag aatgtacaga2881 aaatagcttt tattgagtaa tattacattt catttatact gtagcaatat atttgtaggt2941 atactatgta agggctttaa ataaaagagg tccattaata cttccttata aaaattctag3001 tctgtttcat tactgcccag atgttttaga gataaatatt tatgcagaag gtatttttga3061 agtctccttt tgtctgatag agtttaacag atatttaaat ttagtgctca gaatccacaa3121 gtcacggtct aaacacactt agaatactac agcataaatc tgttagcatt attgccaaat3181 aagacagttg ggatccaaac ccaagtcttg agcaatgttt ttctcaaaaa gctgctatcc3241 aatgatatag gaaaatacat tgtgttttcc taaacacact tttcttttta aatgtgcttc3301 attgtttgat ttggtcctgc ctaaatttca caagctaggc caatgaaggc tgaatcaaag3361 acatttcatc caccaatatc atgtgtagat attatgtata gaaaataaaa taaattatgg3421 ctccaaaaaa aaaaaaaaaa

Other features and advantages of the invention will be apparent to thoseskilled in the art from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D depict characterization of BMPS during differentiation. FIG.1A depicts a diagram of a differentiation protocol. FIG. 1B depicts sizeof aggregates measured during the 3D neuronal differentiation. Negativedays on the x-axis represent 3D cells cultured in NPC medium whilepositive days represent 3D cells cultured in differentiation medium.FIG. 1C1-C5 depicts BMPS mRNA and miRNA expression of different markersduring differentiation. FIG. 1D depicts flow cytometry populationanalysis of BMPS at different stages of differentiation.

FIGS. 2A-2C depict morphological characterization of BMPS. FIG. 2Adepicts co-immunostaining of neurons with markers. MAP2+ neurons wereco-immunostained with the maturation marker Nestin at 2, 4, and 8 weeksof differentiation, which showed progressive increase of MAP2+ neuronsand decrease of Nestin+ cells over time (panels a, b, c), demonstratingneuronal maturation. Co-immunostaining of neurons (NF-H) with the myelinmarker MBP at 2, 4, and 8 weeks of differentiation (d,e,f, respectively)showed progressive increase of MBP+ cells in association with axonalprocesses. An increasing number of MBP+ cells (oligodendrocytes) wasobserved in association with axons (panels d, e, f). FIG. 2B depictsneuronal and glial cell diversity was evaluated at 8 weeks. Neurons(MAP2, NF, SYP and SMI32) were visualized interacting with glia (GFAPand NOGOA). Neurons disclosed characteristic perykaria, dendrites (MAP2,panels a, b) and axons (NF, SMI32, panels c-f) associated with glia.Neurons exhibited diverse neurotransmitter identities shown byidentification of glutamatergic VGLUT1+(panels g, h), GABAergicCALB+(panels i, j) and dopaminergic TH (panels k, 1) neurons. FIG. 2Cdepicts that GFAP+ astroglia and CNPase+, 01+ and MBP+ oligodendrogliawere identified. Oligodendroglia appeared mixed among astrocytes (panelsa, b). 01+(panels c, d) and MBP+(panels e, f) oligodendrocytes wereassociated with axonal processes. Astrocytes established relationshipswith oligodendrocytes and exhibited characteristic multipolar processes(panels g, h). MBP+ oligodendrocytes issued processes in associationwith axons (panel i) 3D-reconstruction demonstrated myelinatingprocesses resembling human myelination (panels j, k). Electronmicroscopy analysis of BMPS at 4 and 8 weeks of differentiationidentified morphology of axonal structures and cells (e.g.,oligodendrocytes) (panel 1). Myelinating-like processes, which closelyresembled cross-sections of myelinated axons of the CNS were identifiedat 8 weeks of differentiation (panel m). FIG. 2D depicts MBP+oligodendrocytes issued processes in close association with axons andseemed to enwrap them at 8 weeks (a,b,c). Myelination calculated as themean percentage MBP positive oligodendrocyte processes coverage ofNF-H-positive axons (a,b,c) at 2, 4 and 8 weeks in at least 2independent experiments showed significant increase of myelinationobserved with time of differentiation (p<0.001) (d). FIG. 2E depicts3D-reconstruction based on confocal z-stacks at 8 weeks demonstrating a“wrapping” myelinating process, which resembled the myelination of axonsin human CNS. FIG. 2F depicts a comparison of expression of neuronal andglial markers at 2 and 8 weeks. At 2 weeks, oligodendrocytes (01,CNPase, NOGOA) were identified without a preferential localization(a,b,e,f,i,j), later they resemble human oligodendrocytes and localizein close proximity with axons (c,d, g,h, k,l). At 2 weeks there are fewMAP2-positive cells without identifiable neuronal shape (l,j) whereas at8 weeks, the MAP2+ cells acquire a well-defined dendritic network (k,l).The amount of astrocytes and density of the astroglial network increaseswith time of differentiation (GFAP, g,h). FIG. 2G depicts variation inthe nuclear morphology. Co-immunostaining of neurons (MAP2) withcell-division marker K167 showed that some cells are dividing (a,b),there was also a small degree of apoptosis demonstrated by positivestaining with CASP3 (c). CASP 3-positive nuclei did not co-localize withmature neurons (d). FIG. 2H depicts ultrastructure analysis by electronmicroscopy of 4 week BMPS showed evidence of cell to cell junctionsdemonstrating functional interactions between the cells (arrows, a,b).Nuclear variation was confirmed by the presence of a few apoptoticnuclei (c) and normal healthy nuclei (d). NF: Neurofilament-heavy-chain,MAP2: Microtubule-associated-protein 2, MBP: myelin-basic-protein,VGLUT1: Vesicular-glutamate-transporter 1, GFAP:Glial-fibrillary-acidic-protein, CALB: Calbindin, NOGOA:Neurite-outgrowth-inhibitor, SYP: Synaptophysin, SMI32:Nonphosphorylated-neurofilament, TH: Tyrosine-hydroxylase, 01: Olig1,CNPase: 2′,3′-Cyclic-nucleotide-3′-phosphodiesterase. Scale Bar: 10 μm.

FIGS. 3A-3F depict electrical activity of BMPS. Cells were cultured in3D for 8 weeks and then cultured in 12-well and 48-well MEA plates for 4more weeks. FIG. 3A depicts heat map recordings from a 48-well plate.FIG. 3B depicts illustration of an active well showing spike morphologyand FIG. 3C depicts spike activity. FIGS. 3D and 3E depictsphase-contrast imaging of the mini-brains on MEAs, electrode diameter is40-50 μm and inter-electrode space is 350 μm. FIG. 3F depicts activitypattern recordings over 0.05 spikes/sec of the electrode over 10 min.

FIGS. 4A-4G depict Parkinson's disease (PD) application of BMPS. BMPSwere differentiated for 4 weeks and exposed to rotenone and MPP+ for 12and 24 hours. FIG. 4A depicts viability (resazurin assay) of BMPS after24 hours rotenone exposure. FIG. 4B depicts ROS (OxiSelect™ In VitroROS/RNS Assay Kit) production of BMPS after 12 and 24 hours rotenoneexposure. FIG. 4C depicts viability (resazurin assay) of BMPS after 24hours MPP+ exposure. FIG. 4D depicts ROS (OxiSelect™ In Vitro ROS/RNSAssay Kit) production of BMPS after 12 and 24 hours MPP+ exposure. FIGS.4E and 4F depict confocal images of BMPS exposed to differentconcentrations of rotenone and MPP+ for NF200 (Red), TH (Green) andHoechst nucleus staining (Blue). FIG. 4G depicts expression of genesassociated with oxidative stress and PD by real time RT-PCR. Graphsrepresent the relative expression of different markers compared tocontrol (cells not treated) after 24 hours exposure to 5 μM rotenone and1 mM MPP+. Genes of interest: mitochondrial complex 5 (ATP50, ATP5C1),mitochondrial complex 1 (NDUFB1), oxidative stress (KEAP1) and genesrelated to PD (TH, SNCA, TBR1, CASP1). Data are presented as mean±SD, of3 independent experiments performed in 3 replicates. *P<0.05 comparingto control (untreated).

FIGS. 5A-5D depict Down's Syndrome application of BMPS. BMPS wereproduced with iPSCs derived from a patient with Down's Syndrome. FIG. 5Adepicts morphological characterization with immunostaining of neurons(MAP2, Synl, TH, SYP), neural precursor cells (nestin) and glial cells(GFAP) at 8 weeks of differentiation. FIG. 5B depicts expression ofgenes in healthy BMPS vs. Down's Syndrome BMPS before and aftertreatment with 5 μM rotenone, after 24 hours exposure. Genes of interestinclude CNS markers (TH, OLIG2, NEFH), mitochondrial markers (ATP5C1,ATPSJ, ATP50) and ROS markers (NFE2L2, SOD1) which were measured bycomparing control with exposed cells to rotenone on both healthy andDown syndrome derived mini-brains. FIGS. 5C and 5D depict karyotyping ofiPSCs derived from the patient with Down's Syndrome. aCGH+SNP resultsfor Down syndrome iPSC line are shown.

FIG. 6 depicts viability of pre-frozen NT2 human teratocarcinoma cellline and iPSC derived mini-brains. Fmedium corresponds to 95% FBS and 5%DMSO. NPC fmedium corresponds to STEMdiff™ Neural Progenitor FreezingMedium. Viability was measured by resazurin cell viability assay.Non-frozen cells at the same stage of differentiation were used ascontrol aggregates.

FIG. 7 depicts an example of a BMPS covered with other cell types.LUHMES fluorescent cells (red) were incorporated to a BMP using gravitysystems to cover the surface of the aggregate.

FIGS. 8A-8E depict morphologic characterization of mature human BMPS.FIG. 8A shows at 8 weeks, neuronal populations exhibited a diversity ofneurotransmitter identities as shown by identification of dopaminergicTH+(a,b), glutamatergic VGLUT1+(c,d) and gabaergic calbindin+(e,f)neurons. Neurons disclosed characteristic axons (NF) and synapticproteins (SYN) (g,h). FIG. 8B depicts two distinctive glial populationswere identified in close interaction with neuronal populations, GFAP+astroglia and CNPase+, 01+, NOGOA+ oligodendroglia. O1+oligodendrocyteswere closely associated with axonal processes (NF) (a,b), CNPase+oligodendroglia appeared mixed among GFAP+ astroglia (c,d) and exhibitedthe characteristic multipolar glial processes, which extended from theperykaria (e,f). NOGOA+ cells were associated with MAP+ neurons (g,h).FIG. 8C depicts example of custom algorithm created using the CellomicsTarget Activation image-analysis software package to study astrocytesand oligodendrocytes (a,b,c,d). Quantification of cell populations as apercentage of the total nuclei count showed 3% NOGOA+ positive cells, 9%CNPase+ cells and 19% GFAP+ cells at 8 weeks (e). FIG. 8D showsCo-expression of mature oligodendroglia markers (MBP and 02). FIG. 8Eshows expression of neuronal markers (VGLUT, TUJ1, SYN). Scale Bar: 10μm.

FIGS. 9A-9D depict the generation of microglia-containing BS (OS)Immortalized Human Microglia—SV40 were incorporated to 7week-differentiated BS by gravity. FIG. 9A shows a diagram of μBSgeneration procedure under one embodiment. FIG. 8B provides a comparisonbetween μBS and BS using different microglia markers (TMEM119, Mertk,Ax1) and hematoxylin/eosin (HE). FIG. 9C provides confocal images ofimmunohistochemistry for the microglia marker IBA1 (green), the neuronalmarker NF200 (red), and nuclear staining (Hoechst 33342, blue) in μBS,48 h after microglia incorporation. FIG. 9D shows details of microgliaaggregations in μBS. Green arrow indicates microglia cluster and redarrow indicates the BS. Bars represent 100 μm (FIG. 9B), 50 μm (FIG. 9C,upper panel), 20 μm (FIG. 9C, all other panels), and 50 μm (FIG. 9D).

FIGS. 10A-10C depicts differences between BS, μBS, and microglia afterLPS treatment. The microglia, BS, μBS, were exposed to 20 ng/mL LPS upto 24 h. FIG. 10A shows gene expression of IL6, IL10, ILlb, CCL2, andTNFa after LPS treatment on the different models (BS, μBS, and microgliacells). Data are shown as fold change of treated versus untreated up toan hour post treatment (3, 6, 12, and 24 h after treatment). FIG. 10Bshows changes in cell cycle in percentage of the cells in G2 plus Sphase in microglia cells, BS and μBS treated or non-treated with LPS.Each dot symbol represent a replicate sample (n+3). FIG. 10C showsAnnexin V Apoptosis Muse assay. Results show % live cells microglia, BS,ad μBS cells treated (+) or not treated (−) with LPS. Each symbolrepresent a replicate (n=3). Statistical analysis in FIG. 10A wasperformed using Dunnett test on ΔΔCt. The asterisks symbols represents*P<0.05 and **P<0.01 (two independent experiments and three biologicalreplicates on each experiment).

FIG. 11 shows virus infection by immunohistochemistry in BrainSphereswith (OS) and without microglia (BS). Green represents the Flavivirusmarker for ZIKV (NS1) and DENV-1, red represents microglia markers IBA1and NF200. Bars represent 50 μm (lower magnification) and 10 μm (highermagnification).

FIGS. 12A-12D depict the effects of Flavivirus infection on 3D humaniPSC-derived brain spheres (BrainSpheres, BS) without or with microgliacells (OS). The microglia cells, BS, and μBS were infected with Dengue,ZIKV-BR, and ZIKV-UG using MOI equal 0.1. FIG. 12A shows the growthkinetics of flaviviruses over time for the three models.

The viral load (RNA copies/mL) are shown with standard deviation. FIG.12B depicts changes in cell cycle shown as percentage of cells in G2/Sphase in BS, μBS, and microglia treated or non-treated with thedifferent viruses. FIG. 12C shows Annexin V Apoptosis Muse assayresults. Results represent % live cells in BS, μBS, and microgliasamples after the different flavivirus treatments. FIG. 12D shows geneexpression relative quantification for TNFα, CCL2, IL-1b, and IL-6 overthe time. Statistical analysis was performed using the Dunnett's Test(*mean equal p<0.05 and **p<0.01) for two independent experiments, threebiological replicates on each experiment.

FIGS. 13A-13C show an alternative protocol performed to incorporatemicroglia into BS 3D cultures. When NPCs reached 95% confluence, 1×10⁶microglia were added to the flask. After 24 h, the co-culture wasdetached mechanically with a cell scraper (Sarstedt, Newton, N.C.,United States, 2-position, Blade 25, 83.1830), re-pipetted fordisaggregation, and counted using the Countess Automated Cell Counter(Invitrogen, Carlsbad, Calif., United States). 2×10⁶ cells per well werethen plated in non-treated 6-well plates and cultured as BS for 8 weeks.The density was chosen to have approximately 10% microglia cells. FIG.13A shows a diagrammatic representation for the alternative protocol.FIG. 13B is immunohistochemistry showing microglia marker (IBA1) andneuronal marker (NF200). FIG. 13C shows immunohistopathologycharacterization of the microglia incorporation for this method of μBSgeneration, markers are mentioned in Example

DETAILED DESCRIPTION

The present invention is based, at least in part, upon the discoverythat brain microphysiological systems (BMPS) can be produced frominduced pluripotent stem cells (iPSCs). Furthermore, the inventionprovides for reproducible BMPS that differentiate into mature neuronsand glial cells (astrocytes and oligodendrocytes) in the central nervoussystem. This model is spontaneously electrophysiological active and maybe reproduced with patient or genetically modified cells. The derivationof 3D BMPS from iPSCs has applications in the study and treatment ofneurological and neurodevelopmental diseases. In some embodiments, thepresent disclosure provides for compositions and methods to study and/ortreat neurodevelopmental and neurodegenerative disorders. In some cases,the neurodevelopmental and neurodegenerative disorders treated and/orstudied by the present disclosure include, but are not limited to,autism, encephalitis, trauma, brain cancer, stroke, Amyotrophic lateralsclerosis, Huntington's Disease, muscular dystrophy, neurodegenerativedisorder, neurodevelopmental disorder, Multiple Sclerosis, infection,Parkinson's Disease and Alzheimer's Disease.

As described herein, the present disclosure provides for the derivationof a multitude of identical brain microphysiological systems (BMPS) fromstem cells, preferably of human origin, but including stem cells fromanimal origin. The preferred starting material are human inducedpluripotent stem cells or embryonic stem cells, although otherpluripotent stem cells such as, for example, neuronal precursor cellsand mesenchymal stem cells may also be employed. Human in-vitro modelsof brain neurophysiology are needed to investigate molecular andcellular mechanisms associated with neurological disorders andneurotoxicity. The techniques herein provide a reproducible iPSC-derivedhuman 3D BMPS that includes differentiated mature neurons and glialcells (astrocytes and oligodendrocytes) that reproduce neuronal-glialinteractions and connectivity. BMPS mature over about eight weeks andshow the critical elements of neuronal function including, but notlimited to, synaptogenesis and neuron-to-neuron (e.g. spontaneouselectric field potentials) and neuronal-glial interactions (e.g.myelination). Advantageously, the BMPS described herein include matureneurons (e.g., glutamatergic, dopaminergic and GABAergic neurons) andglial cells (e.g., astrocytes and oligodendrocytes). Quantification ofthe different cell types exhibited high reproducibility betweenexperiments. Moreover, the BMPS disclosed herein present neuron andglial functions such as spontaneous electrical activity and axonmyelination. The BMPS described herein are able to mimic themicroenvironment of the central nervous system, which is a significantadvance in the field of neurobiology as this ability has not beenachieved at this level of functionality, reproducibility, andconsistency in prior art in vitro systems.

In particular, the high amount of myelination of axons (up to 40%) inthe disclosed BMPS represents a significant improvement over the priorart. Myelin pathology is a rather frequent condition in demyelinatingand inflammatory disorders such as multiple sclerosis and post-infectiondiseases as well as other neurological diseases such as acute andpost-traumatic brain injury, stroke and neurodegenerative disorders (seee.g., Fumagalli et al., 2016; Tse and Herrup, 2016). Moreover, themyelination process can be perturbed by exposure to chemicals and drugs(see e.g., Garcia et al., 2005; Brubaker et al., 2009; Creeley et al.,2013) during brain development and adulthood. For example, the BMPSdisclosed herein show 40% overall myelination after 8 weeks ofdifferentiation. Myelin was observed by immunohistochemistry andconfirmed by confocal microscopy 3D reconstruction and electronmicroscopy. These findings are of particular relevance since myelin iscrucial for proper neuronal function and development. The ability toassess oligodendroglia function and mechanisms associated withmyelination in this BMPS model provide an excellent tool for futurestudies of neurological disorders such as multiple sclerosis and otherdemyelinating diseases. Thus, the BMPS provides a suitable and reliablemodel to investigate neuron-neuroglia function in neurotoxicology orother pathogenic mechanisms that has heretofore not been available inthe prior art.

The method disclosed combines gyratory shaking or regular stirring andthe addition of growth factors to obtain the basic model. Suitableconditions as to how to achieve reproducible brain composition aredisclosed herein. In contrast to earlier models, identical units of BMPSare produced, which allow comparative testing for the purpose of productdevelopment or safety assessments.

According to the techniques herein, a number of additional measurescomplement the basic BMPS to increase their completeness in modeling thehuman brain and improve its usefulness for such testing, for example:

1. The addition of microglia: All stem-cell-derived brain modelsdescribed so far lack micro-glia. The techniques herein provide that theaddition of micro-glia precursor cells and suitable growth factors mayallow microglia to be added to the model. Suitable cells may bemonocytes (e.g., human monocytes), hematopoetic stem cells, respective(pro-)monocyte cell lines, and isolated microglia.

2. The addition of a blood-brain-barrier: The human brain is protectedby a tight blood-brain-barrier that excludes many substances from thebrain. For the first time, the techniques herein provide a method toform a blood-brain-barrier to the BMPS via cells such as, for example,human endothelial cells.

3. Addition of reporter and reporter cells: During the generation of theBMPS, cells carrying reporter for testing purposes may be used or added.These include, but are not limited to, fluorescent or luminescentmarkers to indicate a certain cell lineage or cell response. Genetictransient or permanent transfections are the primary, but not only,method of choice.

4. The BMPS may also be produced, entirely or in its components, fromcells from a specific genetic background, e.g. from patients with aspecific disease or after selective genetic manipulation of the cells.

5. The versatility of the BMPS may be improved by combining it withelectrodes including, but not limited to, micro-electrode arrays (MEA).

6. The versatility of the BMPS may be improved by combining it withother MPS (organ models) platforms such as, for example, microfluidichuman-on-chip systems, perfusion chambers and others.

7. Transportability of BMPS: Methods to cryopreserve BMPS weredeveloped, which allow transport to other laboratories and testing orintegration into multi-MPS platforms.

Simplified neural in vitro systems do not reflect physiology,interactions between different cell types, or human genetics. Inducedpluripotent stem cells (iPSC)-derived human-relevant microphysiologicalsystems (MPS) better mimic the organ level, but are too complex forchemical and drug screening. As described herein, a reproducible 3Dbrain MPS (BMPS) that differentiates into mature neurons and glial cells(astrocytes and oligodendrocytes), which reproduces the topology ofneuronal-glial interactions and connectivity in the central nervoussystem was developed. BMPS from healthy donors or patients evolve from aperiod of differentiation to maturity over about 8 weeks, includingsynaptogenesis, neuron-neuron interactions (e.g. spontaneous electricfield potentials) and neuronal-glial interactions (e.g. myelination ofaxons), which mimic the microenvironment of the central nervous system.Effects of substances on neurodevelopment may be studied during thisphase of BMPS development. In an exemplary embodiment, the techniquesherein were used to study Parkinson's disease (PD) by evaluatingneurotoxicants with a link to PD pathogenesis. Exposure to 5 μM rotenoneor 100 μM 1-methyl-4-phenylpyridinium (MPP+) (or 1 mM1-methyl-4-phenylpyridinium (MPP+) for gene expression studies)disrupted dopaminergic neurons, as observed by immunohistochemistry andaltered expression of PD-related genes (TH, TBR1, SNCA, KEAP1, NDU1-131,ATP5C1, ATP50 and CASP1), thus recapitulating hallmarks of PDpathogenesis linked to toxicant compounds in the respective animalmodels. The BMPS, as described herein, provide a suitable and reliablemodel to investigate neuron-neuroglia function in neurotoxicity or otherpathogenic mechanisms.

There is growing concern about the continuing increase inneurodevelopmental and -degenerative disorders such as autism [1, 2],Parkinson's [3] and Alzheimer disease [4]. Although genetic factors playan important role, environmental factors such as pesticides, airpollution, cigarette smoke, and dietary toxicants appear to contribute[5, 6, 7]. Due to a lack of mechanistic understanding, it is difficultto study their contributions and interactions with respect toneurotoxicity and neurological disorders. The complexity of the CNSmakes it challenging to find appropriate in vitro human-relevant models,ideally from different genetic backgrounds, that are able torecapitulate the relevant pathophysiology. The poor predictive abilityof animal-based models for human health, which may fail to mimic humanpathology as outlined in the costly and time-consuming currentdevelopmental neurotoxicity (DNT) guidelines, contributes to the lack ofreliable information on DNT mechanisms [8]. At the same time, more than90% of all drugs fail clinical trials after extensive animal testing [9]due, in part, to the fact that animal studies often do not reflect humanphysiology and inter-individual differences. Simple in vitro systems donot represent physiology and organ function [10], which creates acritical demand for better models in drug development, study of diseasemechanisms and progression, bioengineering and toxicological testing.

Attempts to generate more complex organotypic cultures ormicrophysiological systems (MPS) [11, 12, 13, 14] have resulted in morephysiological multicellular 3D co-culture models able to simulate afunctional part of the brain [15, 16]. 3D MPS have shown increased cellsurvival, differentiation, cell-cell interactions and can reproduce thecomplexity of the organ more closely [18]. Recent US research programsby NIH, FDA, DARPA, and DTRA have initiated the systematic developmentof MPS, including the model presented here, and their combinations tohuman-on-a-chip technologies to assess the safety and efficacy ofcountermeasures to biological and chemical terrorism and warfare [19].

The discovery of induced pluripotent stem cells (iPSC) and new protocolsto differentiate them into various cell types have boosted thedevelopment of human in vitro models [20, 21]. iPSC from healthy orpatient donors with a specific disease [22, 23, 24, 12] used in MPSpromise more human-representative models, e.g. the brain organoids byLancaster et al. and Kadoshima et al., have been able to recapitulatefeatures of human cortical development [15, 16]. These complex systemspresent novel tools to study biological mechanisms in the CNS, however,they have certain limitations: 1) an elaborate and complex protocol, 2)size differences between organoids, 3) necrosis in the center of theorganoid, 4) low reproducibility in cell differentiation. The human BMPSdescribed herein overcomes these limitations. The reproducible in vitroiPSC-derived human 3D brain microphysiological system (BMPS) iscomprised of differentiated and mature neurons and glial cells(astrocytes and oligodendrocytes).

The techniques herein provide a reproducible BMPS that contains severaldifferent cell types of the human brain, such as glutamatergic,dopaminergic and GABAergic neurons, astrocytes and oligodendrocytes.Moreover, the system has shown neural functionality as observed byspontaneous electrical activity and myelination of axons. Furthermore,the BMPS is reproducible from batch to batch and displays differencesbetween healthy and patient donors. In addition, the obtained resultsdemonstrate the application of such BMPS to the study of neurologicaldisorders such as, for example, Parkinson's Disease (PD).

The brain MPS described herein is a versatile tool for more complextesting platforms and strategies as well as research into neurotoxicity(e.g., developmental), CNS physiology and pathology. Some stemcell-derived brain microphysiological systems have been developed in thelatest years showing the capability to recapitulate some of the in vivobiological process [36, 37, 38]. These models have an enormous advantageover the classical in vitro models to study various differentiationmechanisms, developmental processes and diseases [15]. However, they aremostly based on human embryonic stem cells raising ethical concerns andnot allowing the use of patient cells. Moreover, they requirecomplicated protocols that may reduce the reproducibility of the systemand make it difficult to use in other fields such as chemical and drugscreening. Some of these complex organoids have a large diameter, whichcan lead to extensive cell death, visible in the core of these tissues[15]. This may be due to insufficient diffusion of nutrients and oxygenin these non-vascularized systems, which may generate artifacts intoxicological and disease measurements and make it difficult to studydifferent endpoints in a medium- to high-throughput manner. In addition,it will be challenging to adapt endpoints, established for relativesimple 2D cultures, to such complex models. In the study describedherein, the ability to generate a high number of viable (about 800 perbatch), BMPS that are homogeneous in size (e.g., about 300 μm) and shapeusing iPSC by applying a constant or regular gyratory shaking orstirring technique as described earlier for rat re-aggregating braincell cultures [40] is shown. Control of the size using specific shakerspeed allowed the aggregates to be maintained below 350 μM in diameter(FIG. 1B) and avoid disparate morphology and/or necrosis in the middleof the organoids. Moreover, a spherical homogeneous shape facilitatesfluorescent quantification and further imaging-based endpoints as wellas reproducibility between aggregates. The BMPS had reproducible cellcomposition by immunomorphological quantification, assessment ofimaging-based endpoints and neurophysiological testing.

The 3D differentiation protocol described herein covered stages fromneuronal precursors to different cell types of the mature CNS. After 2weeks, BMPS consisted of an immature population of cells, showingminimal neuronal networks, low percentage of mature astrocytes andoligodendrocytes, with no myelin basic protein expression (FIG. 1C).Cell populations in the BMPS were further differentiated and maturedover time (FIG. 2A). Evidence of iPSC differentiation into mature BMPSwas supported by decreased Nestin expression over time. Nestin isnormally expressed in embryonic tissue and its expression decreases withage in humans, therefore its decrement is a sign of maturation towardsthe adult phenotype [41, 42]. Also, the increasing presence of matureneuronal and glial markers such as MAP2, GFAP, Olig1 and MBP corroboratedifferentiation of the system. Different markers of pluripotency andproliferation decreased during the differentiation process, indicatingmaturing of the in vitro system (FIGS. 1C and 1D). Neuronal precursormarkers such as Nestin, SOX1, SOX2 and the proliferation marker Ki67decreased at the gene expression level and in flow cytometrymeasurements during the differentiation process (FIGS. 1C and 1D). Geneexpression studies, flow cytometry, image analysis, immunostaining andmiRNA studies have demonstrated an increase of cell maturation markers,which follows the BMPS differentiation (FIGS. 1A-1D, 2A-2H and 9A-9C).Obtained data demonstrate that this simple protocol is sufficient togenerate representative CNS cell phenotypes that can reproduce variousstages of differentiation. The presence of GABAergic neurons,dopaminergic neurons and glutamatergic neurons was observed byimmunohistochemistry and real-time-PCR data (FIG. 1C and FIG. 2B). Inaddition, miRNAs such as mir-124, mir-132, mir-128, mir-137 and mir133bwith a role in nervous system differentiation and neuronal degeneration[43, 44] increased during differentiation in patterns consistent withthe in vivo situation. Moreover, the BMPS described herein producedspontaneous electrical activity (FIG. 3) confirming neuronalfunctionality of the system. However, further optimizations of theelectrophysiological measurements using MEAs in 3D systems are needed.

Most of the brain MPS published so far are entirely focused on neuronsand not glia populations [45, 46]; the brain MPS described herein is thefirst 3D model with fully characterized mature human oligodendrocytes,astrocytes and neurons, derived from iPSC. Astrocytes andoligodendrocytes play an important role during neuronal development,plasticity and neuronal injury. Astrocytes have a role in protectingneurons, increasing neuronal viability and mitochondrial biogenesis fromboth exogenous (e.g. chemicals) or endogenous (such as glutamate-inducedexcitotoxicity or the Alzheimer related A131-42) toxicity [47, 48, 49,50]. Astrocytes have an especially important role in neuroprotectionfrom oxidative stress. Oxidative stress is known to be involved in anumber of neuropathological conditions (such as neurodegenerativediseases) [51, 52, 53]. Thus, the presence of astrocytes in a biologicalsystem to study disease is crucial due to their role in detoxificationand neuronal protection. Immunochemistry results from the iPSC-derivedBMPS showed low numbers of astrocytes (GFAP-positive cells) at 2 weeksof differentiation, which increased continuously throughoutdifferentiation (FIG. 2F-2H, and FIG. 2A). Real-time RT-PCR datasupports these findings, as a continuous increase in both s100b and GFAPmRNA levels could be observed from 2 weeks up to 8 weeks old BMPSImmunohistochemistry and RT-PCR data results showed increasing numbersof astrocytes (GFAP-positive cells) in the BMPS model, reaching 19%astrocytes of the total cell population at 8 weeks. After 4 weeks ofdifferentiation, astrocytes demonstrated increased positive staining forGFAP and the presence of glial network was observed (FIG. 2C, panels g,h). At the same time, the presence of oligodendrocytes and myelinationof axons could be observed in the system described herein. This processis highly important, since it is known to be involved in manydegenerative diseases such as multiple sclerosis [54], congenitalhypomyelination [55], progressive multifocal leukoencephalopathy causedby JC virus infection [56], periventricular leukomalacia (PVL) [57] andAlzheimer's disease [58]. Moreover, several chemicals such as ethanol[59], tellurium [60] and lead [(61, 62, 63, 64, 65] have shown to havean effect on the myelination process.

The presence of astroglia and oligodendroglia in the model describedherein brings the system closer to the in vivo brain physiology, whichis a crucial component to study neurodegeneration and neurotoxicity. Inaddition, the system has shown functionality as seen by imaging ofcell-cell junctions, myelination, a rich astroglial network andelectrical activity (FIG. 3). These characteristics make the BMPSdescribed herein a promising tool to study interactions between humanneuronal cells in neurological diseases. The use of iPSCs makes itpossible to study genetic factors and gene/environment interactions.

An assessment of the myelination process by quantification of MBPimmunostaining along axons showed an increase over time reaching 42% ofmyelinated axons at 8 weeks (FIG. 2D). 3D reconstruction of confocalz-stacks images (FIGS. 2C and 2E) and electron microscopy confirmed thewrapping of axonal structures after 8 weeks of differentiation (FIG.2C). These findings are of particular relevance since myelin is acritical element for proper neuronal function and development, theensheathment of axons by myelin allows faster action potentialtransmission, reduces axonal energy consumption and protects the axonsfrom degeneration[79]. Furthermore, recent evidence suggests thatoligodendrocytes and myelin have a role in the metabolic support ofaxons independent of their role in action potential conduction,highlighting their importance in neuronal survival[80]. The ability ofassessing oligodendroglia function and mechanisms associated withmyelination in the BMPS model provide an excellent tool for futurestudies of neurological disorders such as multiple sclerosis and otherdemyelinating disorders.

In one embodiment, the model described herein is useful for studyingParkinson's disease (PD). Traditionally, PD has been described as apre-synaptic degenerative process that affects dopaminergic neurons andinduces a fundamental motor disorder [66], however, non-motor symptomscan also be present [67]. Research in Parkinson's disease isexperiencing an upswing at the moment, owing to a lack of curative drugsfor the large number of patients. Drug testing is nearly exclusivelyperformed in vivo in the so-called MPTP (the parent compound to themetabolite MPP+ used here), rotenone, methamphetamine and6-hydroxydopamine models requiring tens of thousands of animals [68, 69,70]. These model toxins are mainly used in mice and primates (and lessin cell cultures) to model a disease state resembling PD. Human neurons,which would be most relevant, are not usually available and existingcell lines are only very poor substitutes. The model described hereinshows that treatment with MPP+ or rotenone induced specific degenerationof dopaminergic neurons in agreement with Parkinson patients and currentanimal models of the disease (FIGS. 4E and 4F). The BMPS PD model hasshown to recapitulate some of the molecular mechanisms of the humandisease, e.g. increase in ROS production (FIGS. 4B and 3D) and changesin genes related to PD (FIG. 4G). BMPS treated with rotenone or MPP+ haddecreased TH gene expression compared to controls, supporting theresults presented in FIGS. 4E and 4F where the dopaminergic neuronalphenotype is altered after treatment with the two chemicals. TBR1encodes a transcription factor involved in the regulation ofdevelopmental processes. It also plays a role in major neurologicaldiseases such as Alzheimer Disease and PD [71]. This gene wasdown-regulated after treatment with non-cytotoxic concentrations of MPP+and rotenone. At the same time, mRNA levels of SNAC were altered.α-Synucleinopathy (common in Parkinson) is a neurodegenerative disease,which consists of the abnormal accumulation of aggregates ofalpha-synuclein protein in neurons, nerve fibers or glial cells [72].Alpha-synuclein plays regulatory roles such as synaptic maintenance,mitochondrial homeostasis, proteasome function, dopamine metabolism[73]. Reduction of SNCA (the alpha-synuclein encoding gene) aftertreatment with 5 μM rotenone and to a lesser extent after 1 mM MPP+exposure could be explained by the alteration of alpha-synuclein proteinmetabolism. However, it may be that longer exposure times are requiredto produce an increase in gene expression. Caspase-1 (CASP1) expressionincreased significantly after 24 h exposure to 1 μM MPP+. Recently, somestudies have identified human enzyme caspase-1 as the protease thatcleaves α-synuclein in vivo [74]. This cleavage generates α-synucleinfragments that are prone to toxic aggregate formation. Finally, effectsupon genes related with mitochondrial function (such as NDUFB1, ATP5C1and ATP50) were down-regulated, more strongly in BMPS treated with MPP+than rotenone. Changes in NDUFB1, indicate an alteration inmitochondrial function, agreeing with the phenomena already described inParkinson's disease. This downregulation is linked to the increase inKEAP1 expression (oxidative stress marker) after 24 h exposure to 1 mMMPP+. The high variability in some of the genes may be explained by theselective effects of these chemicals (especially MPP+) to dopaminergicneurons, which represent only a subpopulation within the BMPS. Whilerotenone and MPP+ alter gene expression of this cell population, theother populations presented in BMPS appear not to be affected. Furtherstudies using cell sorting could identify cell-specific effects.

This disclosure provides for a description of a brain microphysiologicalsystem aiming to study various aspects of brain development,pathophysiology and disturbance by genetic and environmental factors.The possibilities to study developmental and neurodegenerativedisorders, infections, toxicity and trauma are emerging with such asystem. Furthermore, the potential to use iPSC from different donorsadds a personalized component to these studies. The high reproducibilityand relatively easy protocol, enables future higher throughput testingof chemicals, and drugs and their potential to induce or treat diseases.

Autism

Autism is a highly variable neurodevelopmental disorder that firstappears during infancy or childhood, and generally follows a steadycourse without remission. Patients with autism may be severely impairedin some respects but normal, or even superior, in others. Overt symptomsgradually begin after the age of six months, become established by agetwo or three years, and tend to continue through adulthood, althoughoften in more muted form.

It is distinguished not by a single symptom, but by a characteristictriad of symptoms: impairments in social interaction; impairments incommunication; and restricted interests and repetitive behavior. Otheraspects, such as atypical eating, are also common but are not essentialfor diagnosis. Autism's individual symptoms occur in the generalpopulation and appear not to associate highly, without a sharp lineseparating pathologically severe from common traits.

While autism is highly heritable, researchers suspect both environmentaland genetic factors as causes. In rare cases, autism is stronglyassociated with agents that cause birth defects. Controversies surroundother proposed environmental causes; for example, the vaccine hypotheseshave been disproven. Autism affects information processing in the brainby altering how nerve cells and their synapses connect and organize; howthis occurs is not well understood. It is one of three recognizeddisorders in the autism spectrum (ASDs), the other two being Aspergersyndrome, which lacks delays in cognitive development and language, andpervasive developmental disorder, not otherwise specified (commonlyabbreviated as PDD-NOS), which is diagnosed when the full set ofcriteria for autism or Asperger syndrome are not met.

Globally, autism is estimated to affect 21.7 million people as of 2013.As of 2010, the number of people affected is estimated at about 1-2 per1,000 worldwide. It occurs four to five times more often in boys thangirls. About 1.5% of children in the United States (one in 68) arediagnosed with ASD as of 2014, a 30% increase from one in 88 in 2012.The rate of autism among adults aged 18 years and over in the UnitedKingdom is 1.1%. The number of people diagnosed has been increasingdramatically since the 1980s, partly due to changes in diagnosticpractice and government-subsidized financial incentives for nameddiagnoses; the question of whether actual rates have increased isunresolved.

Autism has a strong genetic basis, although the genetics of autism arecomplex and it is unclear whether ASD is explained more by raremutations with major effects, or by rare multigene interactions ofcommon genetic variants. Complexity arises due to interactions amongmultiple genes, the environment, and epigenetic factors which do notchange DNA but are heritable and influence gene expression. Studies oftwins suggest that heritability is 0.7 for autism and as high as 0.9 forASD, and siblings of those with autism are about 25 times more likely tobe autistic than the general population. However, most of the mutationsthat increase autism risk have not been identified. Typically, autismcannot be traced to a Mendelian (single-gene) mutation or to a singlechromosome abnormality, and none of the genetic syndromes associatedwith ASDs have been shown to selectively cause ASD. Numerous candidategenes have been located, with only small effects attributable to anyparticular gene. The large number of autistic individuals withunaffected family members may result from copy numbervariations—spontaneous deletions or duplications in genetic materialduring meiosis. Hence, a substantial fraction of autism cases may betraceable to genetic causes that are highly heritable but not inherited:that is, the mutation that causes the autism is not present in theparental genome.

Several lines of evidence point to synaptic dysfunction as a cause ofautism. Some rare mutations may lead to autism by disrupting somesynaptic pathways, such as those involved with cell adhesion. Genereplacement studies in mice suggest that autistic symptoms are closelyrelated to later developmental steps that depend on activity in synapsesand on activity-dependent changes. All known teratogens (agents thatcause birth defects) related to the risk of autism appear to act duringthe first eight weeks from conception, and though this does not excludethe possibility that autism can be initiated or affected later, there isstrong evidence that autism arises very early in development.

Exposure to air pollution during pregnancy, especially heavy metals andparticulates, may increase the risk of autism. Environmental factorsthat have been claimed to contribute to or exacerbate autism, or may beimportant in future research, include certain foods, infectiousdiseases, solvents, diesel exhaust, PCBs, phthalates and phenols used inplastic products, pesticides, brominated flame retardants, alcohol,smoking, illicit drugs, vaccines, and prenatal stress, although no linkshave been found, and some have been completely disproven.

Autism does not have a clear unifying mechanism at either the molecular,cellular, or systems level; it is not known whether autism is a fewdisorders caused by mutations converging on a few common molecularpathways, or is (like intellectual disability) a large set of disorderswith diverse mechanisms. Autism appears to result from developmentalfactors that affect many or all functional brain systems, and to disturbthe timing of brain development more than the final product.Neuroanatomical studies and the associations with teratogens stronglysuggest that autism's mechanism includes alteration of brain developmentsoon after conception. This anomaly appears to start a cascade ofpathological events in the brain that are significantly influenced byenvironmental factors. Just after birth, the brains of children withautism tend to grow faster than usual, followed by normal or relativelyslower growth in childhood. It is not known whether early overgrowthoccurs in all children with autism. It seems to be most prominent inbrain areas underlying the development of higher cognitivespecialization. Hypotheses for the cellular and molecular bases ofpathological early overgrowth include the following: an excess ofneurons that causes local over connectivity in key brain regions,disturbed neuronal migration during early gestation, unbalancedexcitatory—inhibitory networks, and abnormal formation of synapses anddendritic spines, for example, by modulation of the neurexing neuroligincell-adhesion system, or by poorly regulated synthesis of synapticproteins.

The immune system is thought to play an important role in autism.Children with autism have been found by researchers to have inflammationof both the peripheral and central immune systems as indicated byincreased levels of pro-inflammatory cytokines and significantactivation of microglia. Biomarkers of abnormal immune function havealso been associated with increased impairments in behaviors that arecharacteristic of the core features of autism such as deficits in socialinteractions and communication. Interactions between the immune systemand the nervous system begin early during the embryonic stage of life,and successful neurodevelopment depends on a balanced immune response.It is thought that activation of a pregnant mother's immune system suchas from environmental toxicants or infection can contribute to causingautism through causing a disruption of brain development. This issupported by recent studies that have found that infection duringpregnancy is associated with an increased risk of autism.

The relationship of neurochemicals to autism is not well understood;several have been investigated, with the most evidence for the role ofserotonin and of genetic differences in its transport. The role of groupI metabotropic glutamate receptors (mGluR) in the pathogenesis offragile X syndrome, the most common identified genetic cause of autism,has led to interest in the possible implications for future autismresearch into this pathway. Some data suggests neuronal overgrowthpotentially related to an increase in several growth hormones or toimpaired regulation of growth factor receptors. Also, some inborn errorsof metabolism are associated with autism, but probably account for lessthan 5% of cases.

The mirror neuron system (MNS) theory of autism hypothesizes thatdistortion in the development of the MNS interferes with imitation andleads to autism's core features of social impairment and communicationdifficulties. The MNS operates when an animal performs an action orobserves another animal perform the same action. The MNS may contributeto an individual's understanding of other people by enabling themodeling of their behavior via embodied simulation of their actions,intentions, and emotions. Several studies have tested this hypothesis bydemonstrating structural abnormalities in MNS regions of individualswith ASD, delay in the activation in the core circuit for imitation inindividuals with Asperger syndrome, and a correlation between reducedMNS activity and severity of the syndrome in children with ASD. However,individuals with autism also have abnormal brain activation in manycircuits outside the MNS and the MNS theory does not explain the normalperformance of children with autism on imitation tasks that involve agoal or object.

The under connectivity theory of autism hypothesizes that autism ismarked by under functioning high-level neural connections andsynchronization, along with an excess of low-level processes. Evidencefor this theory has been found in functional neuroimaging studies onautistic individuals and by a brainwave study that suggested that adultswith ASD have local over connectivity in the cortex and weak functionalconnections between the frontal lobe and the rest of the cortex. Otherevidence suggests the under connectivity is mainly within eachhemisphere of the cortex and that autism is a disorder of theassociation cortex.

From studies based on event-related potentials, transient changes to thebrain's electrical activity in response to stimuli, there isconsiderable evidence for differences in autistic individuals withrespect to attention, orientation to auditory and visual stimuli,novelty detection, language and face processing, and informationstorage; several studies have found a preference for nonsocial stimuli.For example, magnetoencephalography studies have found evidence inchildren with autism of delayed responses in the brain's processing ofauditory signals.

Relations have been found between autism and schizophrenia based onduplications and deletions of chromosomes; research showed thatschizophrenia and autism are significantly more common in combinationwith 1q21.1 deletion syndrome. Research on autism/schizophreniarelations for chromosome 15 (15q13.3), chromosome 16 (16p13.1) andchromosome 17 (17p12) are inconclusive.

Diagnosis is based on behavior, not cause or mechanism. Under the DSM-5,autism is characterized by persistent deficits in social communicationand interaction across multiple contexts, as well as restricted,repetitive patterns of behavior, interests, or activities. Thesedeficits are present in early childhood, typically before age three, andlead to clinically significant functional impairment. Sample symptomsinclude lack of social or emotional reciprocity, stereotyped andrepetitive use of language or idiosyncratic language, and persistentpreoccupation with unusual objects. The disturbance must not be betteraccounted for by Rett syndrome, intellectual disability or globaldevelopmental delay. ICD-10 uses essentially the same definition. Apediatrician commonly performs a preliminary investigation by takingdevelopmental history and physically examining the child. If warranted,diagnosis and evaluations are conducted with help from ASD specialists,observing and assessing cognitive, communication, family, and otherfactors using standardized tools, and taking into account any associatedmedical conditions. A pediatric neuropsychologist is often asked toassess behavior and cognitive skills, both to aid diagnosis and to helprecommend educational interventions.

Clinical genetics evaluations are often done once ASD is diagnosed,particularly when other symptoms already suggest a genetic cause.Although genetic technology allows clinical geneticists to link anestimated 40% of cases to genetic causes, consensus guidelines in the USand UK are limited to high-resolution chromosome and fragile X testing.Metabolic and neuroimaging tests are sometimes helpful, but are notroutine.

Many medications are used to treat ASD symptoms that interfere withintegrating a child into home or school when behavioral treatment fails.More than half of US children diagnosed with ASD are prescribedpsychoactive drugs or anticonvulsants, with the most common drug classesbeing antidepressants, stimulants, and antipsychotics. Antipsychotics,such as risperidone and aripiprazole, have been found to be useful fortreating some conditions associated with autism, including irritability,repetitive behavior, and sleeplessness. A person with ASD may respondatypically to medications, the medications can have adverse effects, andno known medication relieves autism's core symptoms of social andcommunication impairments. Experiments in mice have reversed or reducedsome symptoms related to autism by replacing or modulating genefunction, suggesting the possibility of targeting therapies to specificrare mutations known to cause autism. Although many alternativetherapies and interventions are available, few are supported byscientific studies. Some alternative treatments may place the child atrisk. A 2008 study found that compared to their peers, autistic boyshave significantly thinner bones if on casein-free diets; in 2005,botched chelation therapy killed a five-year-old child with autism.There has been early research looking at hyperbaric treatments inchildren with autism.

Parkinson's Disease

Parkinson's disease (PD, also known as idiopathic or primaryparkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans) isa degenerative disorder of the central nervous system mainly affectingthe motor system. The motor symptoms of Parkinson's disease result fromthe death of dopamine-generating cells in the substantia nigra, a regionof the midbrain. The causes of this cell death are poorly understood.Early in the course of the disease, the most obvious symptoms aremovement-related; these include shaking, rigidity, slowness of movementand difficulty with walking and gait. Later, thinking and behavioralproblems may arise, with dementia commonly occurring in the advancedstages of the disease, and depression is the most common psychiatricsymptom. Other symptoms include sensory, sleep and emotional problems.Parkinson's disease is more common in older people, with most casesoccurring after the age of 50; when it is seen in young adults, it iscalled young onset PD (YOPD).

The main motor symptoms are collectively called “parkinsonism,” or a“parkinsonian syndrome.” The disease can be either primary or secondary.Primary Parkinson's disease is referred to as idiopathic (having noknown cause), although some atypical cases have a genetic origin, whilesecondary parkinsonism is due to known causes like toxins. The pathologyof the disease is characterized by the accumulation of a protein intoLewy bodies in neurons, and insufficient formation and activity ofdopamine in certain parts of the midbrain. Where the Lewy bodies arelocated is often related to the expression and degree of the symptoms ofan individual. Diagnosis of typical cases is mainly based on symptoms,with tests such as neuroimaging being used for confirmation.

Diagnosis of Parkinson's disease involves a physician taking a medicalhistory and performing a neurological examination. There is no lab testthat will clearly identify the disease, but brain scans are sometimesused to rule out disorders that could give rise to similar symptoms.People may be given levodopa and resulting relief of motor impairmenttends to confirm diagnosis. The finding of Lewy bodies in the midbrainon autopsy is usually considered proof that the person had Parkinson'sdisease. The progress of the illness over time may reveal it is notParkinson's disease, and some authorities recommend that the diagnosisbe periodically reviewed. Other causes that can secondarily produce aparkinsonian syndrome are Alzheimer's disease, multiple cerebralinfarction and drug-induced parkinsonism. Parkinson plus syndromes suchas progressive supranuclear palsy and multiple system atrophy must beruled out. Anti-Parkinson's medications are typically less effective atcontrolling symptoms in Parkinson plus syndromes. Faster progressionrates, early cognitive dysfunction or postural instability, minimaltremor or symmetry at onset may indicate a Parkinson plus disease ratherthan PD itself. Genetic forms are usually classified as PD, although theterms familial Parkinson's disease and familial parkinsonism are usedfor disease entities with an autosomal dominant or recessive pattern ofinheritance.

The PD Society Brain Bank criteria require slowness of movement(bradykinesia) plus either rigidity, resting tremor, or posturalinstability. Other possible causes for these symptoms need to be ruledout prior to diagnosis with PD. Finally, three or more of the followingfeatures are required during onset or evolution: unilateral onset,tremor at rest, progression in time, asymmetry of motor symptoms,response to levodopa for at least five years, clinical course of atleast ten years and appearance of dyskinesias induced by the intake ofexcessive levodopa. Accuracy of diagnostic criteria evaluated at autopsyis 75-90%, with specialists such as neurologists having the highestrates. Computed tomography (CT) and conventional magnetic resonanceimaging (MRI) brain scans of people with PD usually appear normal. Thesetechniques are nevertheless useful to rule out other diseases that canbe secondary causes of parkinsonism, such as basal ganglia tumors,vascular pathology and hydrocephalus. A specific technique of MRI,diffusion MRI, has been reported to be useful at discriminating betweentypical and atypical parkinsonism, although its exact diagnostic valueis still under investigation. Dopaminergic function in the basal gangliacan be measured with different PET and SPECT radiotracers. Examples areioflupane (123I) (trade name DaTSCAN) and iometopane (Dopascan) forSPECT or fluorodeoxyglucose (18F) and DTBZ for PET. A pattern of reduceddopaminergic activity in the basal ganglia can aid in diagnosing PD.

Treatments, typically the medications L-DOPA and dopamine agonists,improve the early symptoms of the disease. As the disease progresses anddopaminergic neurons continue to be lost, these drugs eventually becomeineffective at treating the symptoms and at the same time produce acomplication marked by involuntary writhing movements. Surgery and deepbrain stimulation have been used to reduce motor symptoms as a lastresort in severe cases where drugs are ineffective. Although dopaminereplacement alleviates the symptomatic motor dysfunction, itseffectiveness is reduced as the disease progresses, leading tounacceptable side effects such as severe motor fluctuations anddyskinesias. Furthermore, there is no therapy that will halt theprogress of the disease. Moreover, this palliative therapeutic approachdoes not address the underlying mechanisms of the disease.

The term parkinsonism is used for a motor syndrome whose main symptomsare tremor at rest, stiffness, slowing of movement and posturalinstability. Parkinsonian syndromes can be divided into four subtypesaccording to their origin: primary or idiopathic, secondary or acquired,hereditary parkinsonism, and Parkinson plus syndromes or multiple systemdegeneration. Usually classified as a movement disorder, PD also givesrise to several non-motor types of symptoms such as sensory deficits,cognitive difficulties or sleep problems. Parkinson plus diseases areprimary parkinsonisms which present additional features. They includemultiple system atrophy, progressive supranuclear palsy, corticobasaldegeneration and dementia with Lewy bodies.

In terms of pathophysiology, PD is considered a synucleiopathy due to anabnormal accumulation of alpha-synuclein protein in the brain in theform of Lewy bodies, as opposed to other diseases such as Alzheimer'sdisease where the brain accumulates tau protein in the form ofneurofibrillary tangles. Nevertheless, there is clinical andpathological overlap between tauopathies and synucleinopathies. The mosttypical symptom of Alzheimer's disease, dementia, occurs in advancedstages of PD, while it is common to find neurofibrillary tangles inbrains affected by PD. Dementia with Lewy bodies (DLB) is anothersynucleinopathy that has similarities with PD, and especially with thesubset of PD cases with dementia. However, the relationship between PDand DLB is complex and still has to be clarified. They may representparts of a continuum or they may be separate diseases.

Mutations in specific genes have been conclusively shown to cause PD.These genes encode alpha-synuclein (SNCA), parkin (PRKN), leucine-richrepeat kinase 2 (LRRK2 or dardarin), PTEN-induced putative kinase 1(PINK1), DJ-1 and ATP13A2. In most cases, people with these mutationswill develop PD. With the exception of LRRK2, however, they account foronly a small minority of cases of PD. The most extensively studiedPD-related genes are SNCA and LRRK2. Mutations in genes including SNCA,LRRK2 and glucocerebrosidase (GBA) have been found to be risk factorsfor sporadic PD. Mutations in GBA are known to cause Gaucher's disease.Genome-wide association studies, which search for mutated alleles withlow penetrance in sporadic cases, have now yielded many positiveresults.

The role of the SNCA gene is important in PD because the alpha-synucleinprotein is the main component of Lewy bodies. The histopathology(microscopic anatomy) of the substantia nigra and several other brainregions shows neuronal loss and Lewy bodies in many of the remainingnerve cells. Neuronal loss is accompanied by death of astrocytes(star-shaped glial cells) and activation of the microglia (another typeof glial cell). Lewy bodies are a key pathological feature of PD.

Alzheimer's Disease

Alzheimer's disease (AD) accounts for 60% to 70% of cases of dementia.It is a chronic neurodegenerative disease that often starts slowly, butprogressively worsens over time. The most common early symptom isshort-term memory loss. As the disease advances, symptoms includeproblems with language, mood swings, loss of motivation, disorientation,behavioral issues, and poorly managed self-care. Gradually, bodilyfunctions are lost, ultimately leading to death. Although the speed ofprogression can vary, the average life expectancy following diagnosis isthree to nine years. The cause of Alzheimer's disease is poorlyunderstood. About 70% of the risk is believed to be genetic with manygenes involved. Other risk factors include a history of head injuries,hypertension, or depression. The disease process is associated withplaques and tangles in the brain.

Alzheimer's disease is characterized by loss of neurons and synapses inthe cerebral cortex and certain subcortical regions. This loss resultsin gross atrophy of the affected regions, including degeneration in thetemporal lobe and parietal lobe, and parts of the frontal cortex andcingulate gyrus Alzheimer's disease has been hypothesized to be aprotein misfolding disease (proteopathy), caused by accumulation ofabnormally folded A-beta and tau proteins in the brain. Plaques are madeup of small peptides, 39-43 amino acids in length, called beta-amyloid(also written as A-beta or Aβ). Beta-amyloid is a fragment from a largerprotein called amyloid precursor protein (APP), a transmembrane proteinthat penetrates through the neuron's membrane. APP is critical to neurongrowth, survival and post-injury repair. In Alzheimer's disease, anunknown process causes APP to be divided into smaller fragments byenzymes through proteolysis. One of these fragments gives rise tofibrils of beta-amyloid, which form clumps that deposit outside neuronsin dense formations known as senile plaques.

A probable diagnosis is based on the history of the illness andcognitive testing with medical imaging and blood tests to rule out otherpossible causes. Initial symptoms are often mistaken for normal ageing.Examination of brain tissue is needed for a definite diagnosis.Alzheimer's disease is diagnosed through a complete medical assessment.There is no one clinical test that can determine whether a person hasAlzheimer's. Usually several tests are performed to rule out any othercause of dementia. The only definitive method of diagnosis isexamination of brain tissue obtained from a biopsy or autopsy. Tests(such as blood tests and brain imaging) are used to rule out othercauses of dementia-like symptoms. Laboratory tests and screeninginclude: complete blood cell count; electrolyte panel; screeningmetabolic panel; thyroid gland function tests; vitamin B-12 folatelevels; tests for syphilis and, depending on history, for humanimmunodeficiency antibodies; urinalysis; electrocardiogram (ECG); chestX-ray; computerized tomography (CT) head scan; and anelectroencephalogram (EEG). A lumbar puncture may also be informative inthe overall diagnosis.

There are no known medications or supplements that decrease risk ofAlzheimer's. Additionally, no known treatments stop or reverseAlzheimer's progression, although some may temporarily improve symptoms.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES Example 1: Characterization of BMPS by Expression of NeuralSpecific Genes During Differentiation

According to the techniques herein, the BMPS model established hereinfollows a stepwise differentiation protocol (FIG. 1A). In the finalstep, cells were differentiated into various neuronal and glial celltypes during constant gyratory shaking. Briefly, the BMPS wereestablished as follows: cells were differentiated, by addition of B27,GDNF and BDNF and withdrawal of stempro, basic FGF and EGF, intodifferent neuronal and glial cell types with CNS functions duringconstant gyratory shaking. Advantageously, the techniques herein providethat the BMPS that were produced were of a spherical shape and aconsistent size. For example, the BMPS showed spherical shapes andcontrolled sizes that were below 350 μm after 17 days in culture, a sizethat avoids necrosis in the center of the aggregate (FIG. 1B) thatoccurs in larger spheroids (e.g., >350 μm) due to nutrient and oxygendeprivation. Nutrient and oxygen deprivation-induced necrosis couldproduce artifacts in the different endpoints measured, especially indisease and toxicity studies. Five days after initiation of aggregationin NPC medium, spheres were on average 130±5 μm in diameter; the sizeincreased to 300±40 μm during the first 17 days in differentiationmedium. From day 17 onwards size remained constant around 310 μm.Advantageously, this technique significantly increases throughput ofBMPS production by allowing simultaneous production of several batcheswith different conditions. Without the shaking condition, aggregatestend to stick together, grow in different shapes, attach to the bottomand in some point get necrotic in the middle of the sphere. Thus,constant gyratory shaking technology is a suitable method to control theshape and size of BMPS.

In order to characterize different stages of the differentiation andmaturation process, BMPS were collected every week up to 8 weeks ofdifferentiation (FIGS. 1C1-C5). Analysis of different neuronal and glialcell-specific genes by real-time reverse transcription polymerase chainreaction (RT-PCR) was performed to characterize the presence of neurons,astrocytes, oligodendrocytes and neural precursor cells (NPC). NPC areself-renewing and proliferating multi-potent cells able to generatedifferent cell types of the central nervous system. The differentiationof NPC in 3D was initiated by changing the medium to differentiationmedium. Gene expression of the cell proliferation marker Ki67 decreased95% after 2 weeks of differentiation (FIG. 1C1, proliferation and stemcell markers). The remaining Ki67 expression appears to be due to thepresence of a small population of NPC and other proliferating cell typessuch as oligodendrocytes and astrocytes (FIG. 1C2, astroglia andoligodendroglia). Astrocyte-specific genes (S100B and GFAP) showed aconstant increase after two weeks, while, differentiation ofoligodendrocytes was induced later, after six weeks of differentiationas shown by OLIG2 gene expression (FIG. 1C2).

Gene expression of specific neurotransmitters or their receptors wasused to characterize the identity of different neuronal populations andthe differentiation patterns of the human iPSC derived BMPS (FIG. 1C4,neuronal markers; right y-axis relative quantification of GRIN1 andGABRA1; MBP, FOXA2, and SLC1A3). GRIN1 encodes the essential Glutamate[NMDA] receptor subunit zeta-1 [25] was increased at very early stagesof differentiation (one week after induction of differentiation) andcontinued to increase up to 5 weeks when it reached a plateau (FIG.1C4). Similarly, GAD1, a GABAergic neuronal gene marker which encodesthe Glutamate decarboxylase 1, and catalyzes decarboxylation ofglutamate to GABA, showed an increase in expression during the first 4weeks of differentiation, reaching a plateau thereafter (FIG. 1C4). Theexpression of tyrosine hydroxylase (TH) a gene, which identifiesdopaminergic neurons, was observed first after three weeks, showingdelayed differentiation compared to glutamatergic neurons. Theexpression of TH increased constantly thereafter reaching an 86-foldincrease at seven weeks compared to NPC (week 0; FIG. 1C4). GABRA1,which encodes the gamma-aminobutyric acid (GABA) receptor, showed asteady increase of expression after 2 weeks and reached its maximumincrease of a 150-fold change at 8 weeks compared to week 0 (FIG. 1C4).Moreover other markers for specific part of the brain, such as ventralmidbrain neuron marker LMX1A, FOXO1 and FOXA2 (Hedlund et al., 2016;Stott et al., 2013), cerebral cortex marker FOXO4, or markers formyelination CNP and MBP (Li and Richardson, 2008; Agrawal et al., 1994)and L-glutamate transport SLC1A6 (Sery et al., 2015) has been studied(FIG. 1D d). Based on the patterns of expression of neuronal genes, theiPSC-derived BMPS model closely represents the different neuronalpopulations of different cortical and subcortical areas of the humanCNS, suggesting that some of the mechanisms implicated in the earlystages of nervous system development are reflected.

To prove that BMPS can be generated from different IPCs, another healthyline (IPS IMR90) and Down syndrome line (DYP0730) were used (FIG. 1C5).Both lines were able to generate BMPS and differentiated to neurons(MAP2 marker), astrocytes (GFAP marker) and oligodendrocytes (OLIG1marker).

Example 2: Characterization of BMPS by Flow Cytometry Analysis ShowsNeuronal Maturation of the Human Induced Pluripotent Stem Cells OverTime

In order to quantify cell populations in the iPSC-derived BMPS andverify the reproducibility between experiments and batches of the cellline (C1, CRL-2097), flow cytometry was performed using CNS-specificantibodies for identification of neural markers (Table 1). Flowcytometry allowed quantifying 60% of cells with proliferation marker(Ki67) at the NPCs stage (week 0), which was reduced duringdifferentiation down to 9% at 2 weeks, 7% at 4 weeks and 1% at 8 weeks(FIG. 1D), indicating a fast reduction of proliferating cells afterinduction of differentiation. This confirms the gene expression data andindicates a fast reduction of proliferating cells after induction ofdifferentiation. This result was confirmed by further analysis of NPCmarkers such as SOX1, SOX2 and Nestin. SOX1 and SOX2 are known to beinvolved in the maintenance of neural progenitor cell identity. Thenumber of SOX1-, SOX2- and NES-positive (NPC marker) cells in the NPCpopulation (week 0) was 46%, 68% and 60%, respectively. SOX1, SOX2 andNES expression was reduced dramatically with differentiation, showingvery low positive populations at eight weeks (2%, 3% and 2%,respectively). This loss in the NPC population during differentiationwas corroborated by Doublecortin (DCX), a microtubule-associated proteinexpressed in neuroblasts and immature neurons: the number ofDCX-positive cells in NPC (week o) was around 30%, which reduced to 22%at two, 17% at four and 4% at eight weeks, respectively. On the otherhand, the marker for mature neurons, Tuj1 (Neuron-specific class IIIbeta-tubulin) presented the opposite pattern. Analysis showed low levelsof Tuj1-positive cells at the NPC stage (week 0). The expression of thismarker in the cell population increased to 70% after 2 weeks ofdifferentiation and remained constant up to 8 weeks. These flowcytometry experiments indicate differentiation and maturation of theBMPS over time.

Quantification of the cell population in at least three independentexperiments showed low variability between cultures, demonstrating thereproducibility of the system. The variation (standard deviation, SD)between experiments decreased with the cell differentiation process andwas very small at the latest maturation stage (eight weeks); DCX SD0.9%, Ki67 SD 0.2%, SOX1 SD 0.7%, SOX2 SD 1.2%, NES SD 0.7% and Tuj1 SD9.8% (FIG. 1E). These results indicate that after eight weeks ofdifferentiation the cellular composition is similar and shows highreproducibility between different BMPS experiments.

TABLE 1 Gene and miRNAs Taqman Assays. List of the primers used for theexperiments. Catalog Assay ID Assay Type Availability Number Assay NameGene Expression Taqman Primers Hs01060665 TaqMan ® Gene Inventoried4331182 BACT Expression Assay Hs99999901 TaqMan ® Gene Inventoried4331182 18S Expression Assay Hs04187831 TaqMan ® Gene Inventoried4331182 NES Expression Assay Hs01032443 TaqMan ® Gene Inventoried4331182 Ki67 Expression Assay Hs01088112 TaqMan ® Gene Inventoried4331182 PAX6 Expression Assay Hs00909233 TaqMan ® Gene Inventoried4331182 GFAP Expression Assay Hs00300164 TaqMan ® Gene Inventoried4331182 OLIG2 Expression Assay Hs00902901 TaqMan ® Gene Inventoried4331182 S100B Expression Assay Hs00609557 TaqMan ® Gene Inventoried4331182 GRIN1 Expression Assay Hs00165941 TaqMan ® Gene Inventoried4331182 TH Expression Assay Hs00971228 TaqMan ® Gene Inventoried 4331182GABRA1 Expression Assay Hs01065893 TaqMan ® Gene Inventoried 4331182GAD1 Expression Assay Hs00199577 TaqMan ® Gene Inventoried 4331182 SYN1Expression Assay Hs00232429 TaqMan ® Gene Inventoried 4331182 TBR1Expression Assay Hs01003383 TaqMan ® Gene Inventoried 4331182 SNCAExpression Assay Hs01003430 TaqMan ® Gene Inventoried 4331182 KEAP1Expression Assay Hs00929425 TaqMan ® Gene Inventoried 4331182 NDUFB1Expression Assay Hs01101219 TaqMan ® Gene Inventoried 4331182 ATP5C1Expression Assay Hs00919163 TaqMan ® Gene Inventoried 4331182 ATP50Expression Assay Hs00354836 TaqMan ® Gene Inventoried 4331182 CASP1Expression Assay Hs00263981 TaqMan ® Gene Inventoried 4331182 CNPExpression Assay Hs01054576 TaqMan ® Gene Inventoried 4331182 FOXO1Expression Assay Hs00188193 TaqMan ® Gene Inventoried 4331182 SLC1A3Expression Assay Hs00936217 TaqMan ® Gene Inventoried 4331182 FOXO4Expression Assay Hs00892663 TaqMan ® Gene Inventoried 4331182 LMX1AExpression Assay Hs00232764 TaqMan ® Gene Inventoried 4331182 FOXA2Expression Assay miRNA Taqman Assays 1182 TaqMan ® Inventoried 4427975mmu-miR- microRNA Assay 124a 2216 TaqMan ® Inventoried 4427975 hsa-miR-microRNA Assay 128a 457 TaqMan ® Inventoried 4427975 hsa-miR- microRNAAssay 132 2247 TaqMan ® Inventoried 4427975 hsa-miR- microRNA Assay 133b1129 TaqMan ® Inventoried 4427975 mmu-miR- microRNA Assay 137 1094Control miRNA Inventoried 4427975 RNU44 Assay

Example 3: MicroRNAs as Neuronal Differentiation Markers in HumaniPSC-Derived BMPS

MicroRNAs (miRNA), known as posttranscriptional regulators ofdevelopmental timing, have recently been established as markers to studythe differentiation process [26]. Expression of neural-specific miRNAsshowed strong induction of miRNAs involved in neurogenesis (FIG. 1C3,miRNA). mir-124, the most abundant brain miRNA, was strongly induced inthe earlier stages of differentiation, then slightly down-regulated ateight weeks of differentiation. This finding correlates with previousstudies, where mir-124 was shown to promote neuronal lineage commitmentat earlier stages of neural stem cells specification by targetinganti-neuronal factors [26]. mir-128, a modulator of late neuraldifferentiation, was strongly up-regulated after 5 weeks ofdifferentiation. mir-137, the most induced miRNA over time in the systemdescribed herein, is known as a regulator of neural differentiation ofembryonic stem cells (ESCs) [27]. mir-132 and mir-133b which areinvolved in regulation of dopaminergic neuron maturation and function,were induced in week three of differentiation, a finding whichcorrelates with the expression pattern of TH. Moreover, mir-132 isinvolved in dendritic spine formation [28]. These results support theview of a coordinated mechanism of neuronal differentiation as reflectedby the patterns of neuronal gene and miRNA expression and neuronal andneurotransmitter identity.

Example 4: Characterization of Human BMPS by Immunohistochemistry andElectron Microscopy Shows Evidence of Differentiation into Mature BrainCell Types

In order to assess the cellular composition and the process ofmaturation of the cells within the human BMPS, the expression of markersfor different CNS cell populations including neurons and glial cells at2, 4 and 8 weeks of differentiation were evaluated usingimmunohistochemistry and electron microscopy techniques. A reproduciblepattern of expression consistent with maturation of the BMPS towardsmature neural phenotypes was found. After 4 weeks of differentiation,the BMPS showed positive staining for mature neuronal markers such asmicrotubule-associated protein 2 (MAP2), neurofilament-heavy chain (NF,SMI32) and synaptophysin (FIG. 2A, 2B). Furthermore, different neuronalsubtypes in the BMPS including dopaminergic (TH-positive neurons),glutamatergic (VGLUT1-positive neurons) and GABAergic interneurons(calbindin-positive neurons) (FIG. 2B, FIG. 8A) were observed. Moreover,the BMPS matured over time of differentiation as seen by decreasedNES-positive cells (FIG. 2A) and increased cell-cell interactions(neuron-neuron and neuron glia) as subsets of neurons showed severalprocesses, which resembled dendritic and axonal projections (FIG. 8A).

A subset of neuronal cells exhibited immunoreactivity for markers suchas NOGOA, 01, 02, and CNPase (FIG. 8B, panels a-j; FIG. 1C5), whichidentifies the presence of mature oligodendrocytes in the BMPS [31, 33].Automatic image quantification showed that oligodendrocytes (CNPase,NOGOA, and Olig1) comprised 3, 9, and 11% of the total cell population,respectively, at 8 weeks of differentiation (FIG. 8C; FIG. 1C5). Similarto the in vivo physiology, these cells were immunoreactive for myelinbasic protein (MBP) (FIG. 2), which characterizes myelinatingoligodendrocytes [32]. Moreover, they had morphological features ofnormal human oligodendrocytes in vivo and appeared in close contact withneuronal processes (FIG. 8a-b , FIG. 2C, 2D) Similarly, populations ofneuroglia such as astrocytes and oligodendrocytes were identified usingspecific antibody markers. A subset of neuroglial cells exhibitimmunoreactivity for markers such as NOGOA, Olig1 and CNPase (FIG. 2C,panels a-f and 2C, panel i), which identify the presence of matureoligodendrocytes in the BMPS [29, 30, 31, 32]. This pattern ofimmunostaining suggests that oligodendrocytes within the BMPS arefunctional and myelinate axons. Similar to the in vivo physiology, thesecells were also immunoreactive for myelin basic protein (MBP) (FIG. 2Cpanel i and 2C panel j), which characterizes myelinatingoligodendrocytes [33, 30]. These cells had morphological features ofnormal human oligodendrocytes and appeared in close contact with neuronprocesses, which resemble axonal structures (FIG. 2C, panels j-m). Inaddition, a high number of mature astrocytes (FIG. 2Ca, 2Cb, 2Cg, 2Chand 2F) at 4 and 8 weeks of differentiation were observed. Morphometricstudies of neuronal processes identified by immunostaining with NFantibodies and MBP markers were used to estimate the percentage ofmyelinated axons within the BMPS with an average of 4% at 2 weeks, 25%at 4 weeks and 42% at 8 weeks of differentiation (p<0.001) (FIG. 2D).All analyzed BMPS showed similar extent of myelination at the samedifferentiation window. Percentages were calculated as the mean of atleast 18 microscopy fields from at least 3 individual BMPS in 2different experiments. Ultrastructural analysis by electron microscopydemonstrated cell projections, which enwrapped cell processes resemblingaxons after 8 weeks of differentiation (FIG. 2C).

GFAP-positive cells formed numerous cell processes organized in anetwork typical for human astrocyte glial processes in vivo, whichestablished contacts with other glial cells and neurons (FIG. 2Cg, 2Ch,2F, and FIG. 8B). Image quantification revealed 19% of astrocytes in thetotal population (FIG. 8C). Altogether, the patterns of cell morphology,immunostaining and cell-cell interactions shown by neuronal and glialcell populations demonstrates that the BMPS recapitulates the cellulartypes and pattern of interactions seen in the human CNS and is,therefore, considered organotypic.

The morphology of cell nuclei observed by immunocytochemistry andelectron microscopy showed some variation in nuclear morphologyattributed to (i) cell proliferation as seen by positive staining forKi67 and Nestin markers, and (ii) nuclear fragmentation likelyassociated with apoptosis as indicated by caspase 3 staining (FIG. 2G,2H) was observed. These observations were also confirmed by electronmicroscopy studies at 4 and 8 weeks of differentiation (FIG. 2H). Thevariation of nuclei morphology likely reflects the active stages of celldifferentiation that BMPS exhibited during all stages of development.The presence of apoptotic nuclei likely resemble stages of cell deathseen in normal neurodevelopment [34, 35]. Importantly, Caspase3-positive nuclei did not concentrate in the center of the spheres andBMPS did not present necrosis in the center of the 3D structures (FIG.2G). Thus, Caspase3-positive nuclei do not appear linked to deprivationof oxygen or nutrients. Caspase has been quantified at eight weeks inBMPS (FIG. 8C). Additionally, FIGS. 8D and 8E depict co-expression ofmature oligodendroglia markers (MBP and 02) and expression of neuronalmarkers (VGLUT, TUJ1, SYN), respectively.

Further analysis of neuronal cell populations and morphology presented apattern of evolution that suggests BMPS maturation as seen byNestin-positive cells decreasing over time of differentiation while MBPexpressing cells increased (FIG. 2A). There was also evidence ofcell-cell interactions as subsets of neurons showed several processes,which resemble dendritic and axonal projections that interact with otherneurons as well as glial cells (FIG. 2B, FIG. 2H). Furthermore, cellsimmunostained with myelin binding protein (MBP) antibodies issuedprojections, which appear to enwrap neuronal processes, which resembleaxons (FIG. 2C, panels i-k, 2C, panel m). The pattern of immunostainingwith MBP and its association with neuronal processes suggests thatoligodendrocytes within the BMPS exhibit myelinating properties such asin the human CNS in vivo. Ultrastructural analysis by electronmicroscopy demonstrated cell projections, which enwrapped cell processesresembling axons (FIG. 2C, panel m).

Example 5: Microelectrode Array Recording of Spontaneous ElectricalActivity of BMPS

To test the neurophysiological properties of the cells within the BMPSmodel, spontaneous electrical activity in BMPS was analyzed bymicro-electrode array (MEA)(see FIG. 3 generally). BMPS were plated in12-well or 48-well MEA plates at 8 weeks of differentiation. Theaggregates were attached to the MEAs using Matrigel coating. Spontaneouselectrical activity was measured starting one week after plating up totwo weeks. The activity was measured for 20 minutes on 7 different days.Electrodes were considered active when the recorded activity was above0.05 spikes/sec. FIG. 3A shows a representative heatmap of a 48-well MEAplate measurement from one 20 minute recording. The heatmap representsthe spike amplitude (μV) with a minimum of 0 μV and maximum of 40 μV(FIG. 3A). The spikes showed a common waveform between differentelectrodes and measurements (FIG. 3B) and neurons were repeatedlyfiring. 25 electrodes, distributed over 19 wells, were included afterthe first step of data analysis. 20 to 40% of these 25 electrodesreached the threshold of 0.05 spikes/sec during each recording. FIG. 3Fshows the spike events of active electrodes from one representative 20minutes recording. These data show potential for the use of MEA tomeasure electrical activity of the 3D BMPS. Further optimization of theprotocol may increase the measurement of the neuronal activity on theelectrodes.

Example 6: A Human 3D Model to Study Parkinson's Disease

Due to the presence of TH-positive dopaminergic neurons in theiPSC-derived BMPS (FIG. 2B, panels k, 1, and FIG. 8), the possibility ofusing this model to study Parkinson's Disease (PD), a neurodegenerativedisorder known to specifically affect dopaminergic neurons, was furtherexplored. Two well-known neurotoxicants, which induce pathogenicprocesses resembling the mechanism associated with neurodegeneration inPD: the illicit drug MPTP's toxic metabolite MPP+ and the broadly usedpesticide rotenone, were selected. Both MPP+ and rotenone interfere withoxidative phosphorylation in mitochondria by inhibiting complex I [36].Initially, cytotoxicity experiments were performed to estimatesub-cytotoxic concentrations of these two compounds affecting onlydopaminergic neurons (FIGS. 4A and 4C). Selective disruption ofdopaminergic neurons but not of any other cell types in the systemsdescribed herein were observed with immunohistochemistry after exposureto 1 μM rotenone and 100 μM MPP+ for 24 h (FIGS. 4E and 4F). This effectwas likely selective even at cytotoxic concentrations of 10 μM rotenoneand 1000 μM MPP+ as these concentrations did not show any alterations inother neurofilament 200-positive neurons. Lower concentrations of thesecompounds may induce effects in dopaminergic neurons, however, theeffect was not as obvious by immunocytochemistry. Higher concentrationsof rotenone and MPP+(up to 50 μM and 5000 μM, respectively) led togeneral cytotoxicity and affected also other neuronal types stainedpositive for neurofilament 200 (FIGS. 4E and F). 5 μM of rotenone and1000 μM of MPP+ were selected for further studies as theseconcentrations induced clear and selective dopaminergic effects.Reactive oxygen species (ROS) were measured in the cellular medium usingthe OxiSelect™ In Vitro ROS/RNS Assay Kit (Cellbiolabs, San Diego,Calif.) as an indication of oxidative stress. Exposure to rotenone at 5μM and MPP+at 1000 μM showed an increase in ROS production after 24hours exposure, while 12 hours showed no statistically significantchanges. Real time RT-PCR was performed in order to determine effects ofboth chemicals on genes related to PD, mitochondrial dysfunction andoxidative stress. Tyrosine hydroxylase (TH, Dopaminergic neuronalmarker) mRNA expression decreased by 84%±11 after exposure to 5 μMrotenone and 70%±9 after exposure to 1000 μM MPP+ for 24 hours.Additional genes related to PD also showed changes at sub-cytotoxicconcentrations of MPP+ and rotenone. The expression of genes that encodeT-box brain 1 (TBR1) and Alpha-synuclein (SNCA) protein decreased after24 hours exposure. The reduction of TBR1 was 70±13% (rotenone) and76±22% (MPP+) and the reduction of SNCA was 72±6% (rotenone) and 41±40%(MPP, however, BMPS exposed to 1 mM MPP+ led to no statisticallysignificant changes in SNCA expression). Expression of genes related tomitochondrial function complex I (NDUFB1) or complex 0 (ATP5C1 or ATP50)tended to decrease in expression but these changes were notstatistically significant. Caspase-1 gene expression, which has beenrelated to SNCA, increased after 24 hours exposure to MPP+. Theseresults demonstrate the potential of BMPS for studies elucidatingmolecular mechanisms of PD, lending itself to PD drug and neurotoxicityscreening.

Example 7: Addition of Microglia

Peripheral blood mononuclear cells (PBMCs) are isolated from fresh orcommercially available cryo-preserved whole blood of pooled healthydonors by Ficoll or Percoll gradient centrifugation. Monocytepopulations are obtained by negative magnet-antibody selection afterFicoll or Percoll gradient and then re-suspend in RPMI 1640. Monocytesare cultured in macrophage serum-free medium, stimulated with a cocktailof cytokines, GM-CSF and IL-34. Monocytes may also be obtained bydifferentiation of iPSCs, hematopoetic or other stem cells. Themicroglia-like cells are combined with neuronal precursor cells inshaker cultures to preferably arrive at a final concentration of 5-8%microglia.

Primary monocytes or iPSC-derived monocytes may be incorporated into thesystem, both at early and later stages of BMPS differentiation. For theearly stages, a number of 2×10⁶ NPCs mixed with 2×10⁴ monocytes areplated per 1 well (6 well-plate). Gyratory shaking is used at 88 rpms togenerate spheres. After 2 days media are replaced with ½ CNSdifferentiation medial (Neurobasal® electro Medium (Gibco) supplementedwith 5% B-27® Electrophysiology (Gibco), 1% glutamax (Gibco), 10 μghuman recombinant GDNF (Gemini), 10 μg human recombinant BDNF (Gemini))and ½ macrophage differentiation media (Dulbecco's modified Eagle'smedium (Invitrogen) supplemented with 10% FCS, 0.055 mMβ-mercaptoethanol, M-CSF (50 ng/ml), and IL-3 (25 ng/ml) (R&D Systems).The medium is replaced every 3 days.

Monocytes can also be incorporated after BMPS differentiation. For that,BMPS are differentiated up to 8 weeks. BMPS spheres are separated in 500μl Eppendorf tubes. 2×10⁴ monocytes are added to the Eppendorf with theBMPS. Tubes are shaking manually every hour, up to 8 hours. After that,BMPS-monocytes are collected and plated in 6 well plates. Cells are kepton constant shaking until use.

The characterization of the immune-competent human organoids can becarried out by immunocytochemically assessing the presence of markerssuch as HLA-DR, and the ionized calcium-binding adapter molecule 1(Iba1), specific microglial markers. Measures of cytokines andchemokines release and expression of receptors associated with microgliafunction (e.g., CCL2 and CX3CL) demonstrates successful engrafting ofthe microglia cells. This modified model is more suitable to investigatethe neuroimmunological component associated with many substanceexposures and diseases.

Example 8: Addition of a Blood Brain Barrier

The blood brain barrier (BBB) has a crucial role in neurotoxicity, beingthe last barrier for substances before reaching the brain. Moreover, theBBB is the bottleneck in brain drug development and is the single mostimportant factor limiting the future growth of neurotherapeutics [81].Most of the in vitro models do not incorporate BBB.

Human brain microvascular endothelial cells (hBMECs) from human iPSCsare incorporated into the BMPS by two techniques. In the first approach,mature BBB endothelial cells and neuronal precursors cells (NPCs) arecombined in a single cells suspension in a ratio of 1:5, gyratoryshaking or stirring are used to generated spheroids and aggregates arecultured up to 8 weeks. In the second technique, mature BMPS (8 weeks ofdifferentiation) are covered by BBB endothelial cells using gravitysystems (aggrewell, gravity well or hanging drops). Cells may be coveredas well with other cell types, such as fluorescent LUHMES cells (FIG.7).

Example 9: Addition of Reporters

The BMPS gives the opportunity to develop cell-based assays allowing forhigh-content imaging (HCI) that can be adapted to high-throughputplatforms, to evaluate the effects of toxicants on key cellularprocesses of neural development and physiology in the culture system.

Example of establishing fluorescent iPSC cell line: Creation of reportercells lines greatly assists imaging efforts by allowing us to avoidcomplications associated with staining 3D cultures, to image subsets ofcells, and to perform functional assays. Differentiated 3D aggregatesfrom iPSC cultures spiked with 1-2% of iPSCs ubiquitously expressingfluorescent protein allow visualizing individual cells within theaggregates aiding quantification of phenotypic parameters, includingneurite outgrowth and migration. Lines expressing markers allowmeasurement of synapse formation (PSD95, Synapsin 1), proliferation(Ki67), glial maturation (GFAP), and calcium signaling (GCaMP).Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR)were used to create the various lines. Similar in function to thewell-established zinc-finger (ZFNs) and TALEN nucleases, the Cas9-CRISPRsystem is a new entrant into the rapidly emerging field of genomeengineering and has been quickly adopted and validated across a widearray of human stem cells. Gene-editing in hiPSCs has traditionally beena technically difficult task but with these advances it is now possibleto generate reporter and mutant cell lines with genetically matchedcontrols [83, 84, 85, 86]; essential tools not only for this project butalso for the future success of using human iPSC-derived cells inquantitative live-cell phenotypic assays of toxicant testing.

Using the CRISPR-Cas9 system, fluorescent protein (FxP) reporter celllines were generated by generating gRNAs targeting the gene ofinterested. In this system as described herein, an RNA guided Cas9endonuclease is used in conjunction with customizable small guide RNAs(gRNAs) to target and cleave any DNA template with a GN21GG sequence;the first G is for the U6 polymerase promoter while the N21GG is for theprotospacer adjacent motif (PAM) sequence requirement of Cas9 [86, 87,89].

For reporter cell generation, homology-directed repair (HDR) guides theinsertion of the appropriate DNA donor fragment into a target site atregions of homology between the donor fragment and the genomic DNAtarget. An ES line that ubiquitously expresses GFP was created byintroducing CAG promoter-driven GFP into the AAVS1 safe harbor locus,and can use these constructs to transfect iPSC cells. For otherreporters, constructs may be created that will direct the integration ofa self-cleaving P2A peptide sequence [90] targeted fluorescent proteincassette in frame at the stop codon of the gene of interest. The P2Asequence engineered between the C-terminus of the endogenous protein andthe fluorescent protein may minimize possible fusion protein functionaldefects. Plasmids encoding the Cas9 nuclease, the targeting gRNA, andappropriate donor DNA will be introduced by electroporation, recombinanthiPSC clones will be manually selected and screened for the desiredinsertion by PCR, and the genotype may be verified by sequencing.Reporter hiPSCs will be subjected to a differentiation protocol andexpression of the reporter validated by examining expression patternsand through immunohistochemistry experiments where it may be determinedwhether the FxP expressing cells co-label with known markers.

Example 10: Using Cells with Specific Genetic Backgrounds

The use of iPSCs, as described herein, has created new opportunities tostudy human diseases and gene/environment interaction [20, 21].Fibroblasts or other somatic cells from healthy and diseased individualscan be reprogrammed into iPSCs, and subsequently be differentiated intoall neural cell types. Similarly, iPSC can be genetically modifiedbefore creating the BMPS. As a proof-of-principle, iPSCs were obtainedfrom patients with Down's syndrome (FIGS. 1C5 and 5A-D), Rett Syndromeand from individuals with mutations in disrupted in schizophrenia 1(DISCI). DISCI may have some functional overlap with TSC-iPSCs as bothare involved in the mTOR cell signaling pathway.

The Down's syndrome model is further characterized (see FIGS. 5A-5D).Down's syndrome iPSCs have been successfully differentiated into neuralprecursor cells (NPCs). Currently the cells are differentiated in 3D andcharacterization by gene expression and immunohistochemistry is beingperformed. The Down's syndrome model has been exposed to compounds thatinduce oxidative stress (rotenone and paraquat). The response wascompared to the model from healthy donors, which were more sensitive tothese compounds than the healthy model.

Example 11: Combining the BMPS with Other Organoids

In some embodiments, BMPS may be combined with other organs and/or organmodel systems. Several groups have been developing organ-on-a-chipplatforms for different organs by using microfluidic techniques. Thoseplatforms are designed to mimic in-vivo fluidic flows in the organs byseparating cell culture chambers and perfusion channels, andsuccessfully demonstrate recapitulation of iPSC-based organ functions.Together with other organ models on these platforms, the BMPS can beintegrated, which allow us to untwine the complex toxicology from organinteractions. Such platforms allow (1) in-situ and high-throughputproduction of mini-brains on chip, (2) in-vivo like fluidic flow aroundmini-brains with enough supply of nutrient and small molecule throughdiffusion, (3) a large number of parallel test of toxic materials, and(4) a real-time monitoring of electrophysiological activities from BMPSwith integrated electrodes. Companies such as TissUse GmbH have designedmicrofluidics platform that allow culture of floating spheres like theBMPS as described herein.

Example 12: Cryopreservation and Other Modes of Transportability

In order to e.g. incorporate the BMPS into platforms or enable any usein other laboratories, transportability of the system was optimized.Preliminary studies have shown possible recovery of the neuronal 3Daggregates after cryopreservation (FIG. 6). A human embryonal carcinomastem cell line, (hNT2), and iPSC derived-aggregates were differentiatedinto mature neurons (8 weeks of differentiation for each cell line) andthen cryopreserved with regular cryopreservation medium (95% FBS and 5%DMSO) or STEMdiff™ Neural Progenitor Freezing Medium (Stem cellstechnologies). After 2 days in liquid nitrogen, cells were thawed.Freezing media was removed and fresh media was added. One day later,viability was measured using the resazurin cell viability assay. hNT2aggregates presented a 70% decrease in viability in both freezing mediaswhile iPSC derived mini-brains showed a 20%-35% reduction in viability(FIG. 6). However, viability recovery of the 3D aggregates is currentlyoptimized using other viability and functional assays. Optimization ofthis protocol will vary additives (DMSO, HES, glycerol, serum etc.), thecooling temperature gradient as well as thawing protocol.

Human iPSC derived mini-brains are kept in culture at 37° C. In order totransport the live mini-brains, temperature must be controlled.Different methods can be used to control temperature during transport.Heating pads combined with an insulated box have been used to transportlive biological material. Disposable chemical pads employ a one-timeexothermic chemical reaction such as catalyzed rusting of iron, ordissolving calcium chloride. The most common reusable heat pads arebased on a chemical reaction that transforms a liquid into a solid thusreleasing energy. Some new heating pads (such as Deltaphase IsothermalPad 3SET, from Braintree Scientific, Inc.) have been able to maintain37° C. for more than 6 hours. 3D mini-brains cultured up to 8 weeks aresent in an insulated material box with heating pads. After transport,viability may be measured.

Example 13: Overview

The techniques herein provide a human BMPS model that is a versatiletool for more complex testing platforms, as well as for research intoCNS physiology, mechanisms associated with (developmental)neurotoxicity, and pathogenesis of neurological disorders. Prior artstem cell-derived brain model systems developed in the past few yearshave shown the capability to recapitulate some of the in vivo biologicalprocesses (Juraver-Geslin and Durand, 2015; Nakano et al., 2012; Krug etal., 2014) and have an advantage over other classical in vitro models asthey facilitate the study of various differentiation mechanisms,developmental processes and diseases (Lancaster et al., 2013).Unfortunately, these prior art systems require complicated protocolsthat reduce the reproducibility of the system and make it difficult touse in other fields such as chemical toxicity and drug screening.Additionally, these prior art models are also limited by largediameters, which lead to extensive cell death in the interior regionsdue to insufficient diffusion of oxygen and nutrients (Lancaster et al.,2013) and other artifacts.

The techniques herein overcome the limitations of the prior art bydeveloping a human in vitro model by the gyratory shaking technique thatenables reliably generation of a high number (about 500 per six-wellplate) of viable BMPS that are homogeneous in size and shape. Control ofsize makes it possible to keep cell aggregates below 350 μM in diameter(FIG. 1C) and thereby avoid disparate morphology and/or necrosis in thecenter of the spheres. Moreover, the BMPS showed reproducible cellcomposition by immunomorphological quantification, assessment ofimaging-based endpoints and flow cytometry analysis.

As described herein, the 3D differentiation protocol for the BMPS coversstages from neuronal precursors to different cell types of the matureCNS. As discussed in detail above, at two weeks, BMPS consisted of animmature population of cells, showing minimal neuronal networks, a lowpercentage of mature astrocytes and oligodendrocytes, and minimal butearly stages of myelin basic protein (MBP) expression. iPSCdifferentiation into mature BMPS was indicated by decreasing NESexpression over time and a progressive expression of mature neuronal andglial markers such as MAP2, GFAP, 01 and MBP. Gene expression studies,flow cytometry, image analysis, immunostaining and miRNA studies haveshown increase of cell maturation markers, which follow the BMPSdifferentiation. The presence of GABAergic neurons, dopaminergic neuronsand glutamatergic neurons was documented by immunohistochemistry andreal-time PCR data. Moreover, the BMPS showed spontaneous electricalactivity, indicating neuronal functionality of the system.

Since astrocytes and oligodendrocytes play important roles duringneuronal development, plasticity and injury, the presence of glial cellpopulations in the presently disclosed BMPS model provides an excellentopportunity for the evaluation of neuronal-glial interactions and therole of glia in pathogenesis and toxicity processes. Astrocytes have animportant role in protecting neurons, increasing neuronal viability andmitochondrial biogenesis from both exogenous (e.g. chemicals) andendogenous toxicity (Shinozaki et al., 2014; Aguirre-Rueda et al.,2015), especially against oxidative stress (Shao et al., 1997; Schwaband McGeer, 2008). Thus, their presence in a biological system to studydisease and neurotoxicity is crucial Immunohistochemistry and RT-PCRresults showed increasing numbers of astrocytes (GFAP-positive cells) inthe BMPS model reaching 19% astrocytes of the total cell population ateight weeks, which is earlier than in previously described corticalspheroids, where similar proportions of GFAP-positive cells wereobserved first at day 181, at day 86 the number of GFAP+ cells was below10% (Pasca et al., 2015).

The most novel element of this BMPS is the presence of mature humanoligodendrocytes with myelination properties, which has not beenachieved in the prior art. Immunocytochemical and ultrastructuralstudies confirmed the morphological identity of these cells (FIG. 2D) asmultiple markers for mature oligodendrocytes were expressed by roundedcells with branching processes and membrane sheaths that are similar tothe ones found in humans in vivo. The structure and morphology wasfurther confirmed by electron microscopy. Quantitative assessment of themyelination process of MBP immunostaining along axons showed an increaseover time of differentiation reaching 42% of myelinated axons at eightweeks (FIG. 2D). 3D reconstruction of confocal z-stacks images (FIG. 2A)and electron microscopy confirmed the wrapping of axonal structuresafter eight weeks of differentiation (FIG. 2C). These findings are ofparticular relevance since myelin is a critical element for properneuronal function and development, and the covering of axons by myelinallows faster action potential transmission, reduces axonal energyconsumption and protects the axons from degeneration (Nave, 2010).Furthermore, recent evidence suggests that oligodendrocytes and myelinhave a role in the metabolic support of axons independent of their rolein action potential conduction, highlighting their importance inneuronal survival (Saab et al., 2013). This is the first time that a 3Dhuman microphysiological system, consisting of different types ofneurons and glial cells, has achieved such a high percentage ofmyelination. The ability to assess oligodendroglia function andmechanisms associated with myelination in this BMPS model provides anexcellent tool for future studies of neurological disorders such asmultiple sclerosis and other demyelinating disorders. As an illustrationit was recently discovered that astroglia cells could promoteoligodendrogenesis via secreted molecules (Jiang et al., 2016). A humanBMPS that consist of neurons, astrocytes and oligodendrocytes isessential to evaluate this mechanism further and to develop a potentialtherapy for demyelinating disorders.

In conclusion, the techniques herein provide a BMPS that replicatescrucial aspects of brain physiology and functionality. The potential forstudying developmental and neurodegenerative disorders, braininfections, toxicity and trauma with such a system is growing.Furthermore, the potential to use iPSCs from different donors adds apersonalized component to these studies. The high reproducibility andrelatively simple protocol, enables future medium-throughput (96-wellformat) testing of chemicals, drugs and their potential to induce ortreat diseases.

Methods and Materials

Chemicals

Rotenone and MPP+ were supplied from Sigma-Aldrich (St. Louis, Mo.). A10 mM rotenone stock was prepared in DMSO Hybri-Max (Sigma) while MPP+was diluted in water to a concentration of 30 mM.

iPSC Generation

CCD1079Sk (ATCC® CRL2097™), IPS IMR90 (WiCELL) and ATCCDYP0730 Human(IPS) Cells (ATCC® ACS1003™) fibroblasts were originally purchased fromATCC. All studies followed institutional IRB protocols approved by theJohns Hopkins University School of Medicine. Human fibroblasts and mouseembryonic fibroblasts (MEFs) were cultured in Dulbecco's modifiedEagle's medium (DMEM, Mediatech Inc.) supplemented with 10% fetal bovineserum (FBS, HyClone) and 2 mM L-glutamine (Invitrogen). MEFs werederived from E13.5 CF-1 mouse embryos. Human iPCS cells were generatedwith the EBV-based vectors as previously described [75]. iPSC from othersources were used as well. Colonies of iPSCs were manually picked after3-6 weeks for further expansion and characterization. iPSCs (passage≤20) were cultured on irradiated MEFs in human embryonic stem cell(hESC) medium comprising D-MEM/F12 (Invitrogen), 20% Knockout SerumReplacement (KSR, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μM MEMNEAA (Invitrogen), 100 μM β-mercaptoethanol (Invitrogen), and 10 ng/mLhuman basic FGF (bFGF, PeproTech). Media were changed daily and iPSClines were passaged using collagenase (Invitrogen, 1 mg/ml in D-MEM/F12for 1 hr at 37° C.). These iPSC lines have been previously fullycharacterized [75].

Neuronal Progenitor Cells (NPC) Production

NPC generated followed the previous published protocol [75]. Briefly,iPSCs colonies were detached from the feeder layer with collagenase (1mg/ml) treatment for 1 hr and suspended in EB medium, comprising ofFGF-2-free hESC medium supplemented with Dorsomorphin (2 μM) and A-83 (2μM), in non-treated polystyrene plates for 4 days with a daily mediumchange. After 4 days, EB medium was replaced by neural induction medium(hNPC medium) comprising of DMEM/F12, N2 supplement, NEAA, heparin (2μg/ml) for 15 more days. The floating neurospheres were then dissociatedto single cells in Accutase and plated in 175 mm flasks and were allowedto expand for 7 days. NPCs were expanded in poly-1-ornithine andlaminin-coated 175 mm flask on StemPro® NSC SFM (Life Technologies).Half of the media was changed every day. Cultures were maintained at 37°C. in an atmosphere of 5% CO₂. After NPC generation, iPSCs colonies weredetached and NPCs were expanded in poly-1-ornithine and laminin-coated175 mm flask in StemPro® NSC SFM (Life Technologies). Half of the mediawas changed every day. Cultures were maintained at 37° C. in anatmosphere of 5% CO2.

BMPS Differentiation

At 100% confluence NPCs were detached mechanically and counted. 2×10⁶cells per well were plated in 2 ml of medium in non-treated 6well-plates. Cells were grown in NPC media for two days under constantgyratory shaking. Subsequently, medium was changed to differentiationmedium (Neurobasal® electro Medium (Gibco) supplemented with 5% B-27®Electrophysiology (Gibco), 1% glutamax (Gibco), 0.02 μg/ml humanrecombinant GDNF (Gemini), 0.02 μg/ml human recombinant BDNF (Gemini))Cultures were maintained at 37° C., 5% CO₂ under constant gyratoryshaking for up to 8 weeks. Differentiation medium was routinely changedevery 2 days.

Size Measurement

Aggregates (n=20) from 3 independent experiments were randomly selectedper time point for obtaining pictures and measuring size using SPOTsoftware 5.0. Results were expressed as mean±SD. Cells were kept twodays in NPC medium, indicated as NPC med. 2 d in FIG. 1B.

RNA and miRNA Extraction

Total RNA was extracted from aggregates every week up to 8 weeks ofdifferentiation using Tripure (Roche, Switzerland) according toChomczynski and Sacchi (1987) [76]. The same RNA extraction method wasused to isolate RNA after toxicant treatment. RNA quantity and puritywas determined using NanoDrop 2000c (Thermo Scientific). One microgramof RNA was reverse-transcribed using the M-MI V Promega ReverseTranscriptase (Promega) according to the manufacturer's recommendations.For miRNA reverse-transcription 60 ng of RNA were reverse transcribedusing TaqMan microRNA Reverse transcription kit in combination withmiRNA specific stem-loop primers, which are a part of TaqMAn microRNAexpression assay. Upto eight stem-loop primers were multiplexed in onereaction.

Quantitative RT-PCR

The expression of genes was evaluated using specific Taqman® geneexpression assays (Life Technologies). miRNA expression was analyzedusing TaqMAn microRNA expression assay in combination with TaqMan miRNAReverse Transcription kit using protocol described in [77]. Table 1shows a summary of the genes assayed. Real time RT-PCRs were performedusing a 7500 Fast Real Time system machine (Applied Biosystems). Foldchanges were calculated using the 2(−ΔΔCt) method [78]. β-actin and 18 swere used as a housekeeping genes for mRNA and RNU44 for miRNA. Therewere no statistically significant differences in expression for β-actin,18s, and RNU44. Data were presented as mean±SD, normalized tohousekeeping genes and week 0.

Immunocytochemistry of the BMPS

BMPS aggregates were collected at 2, 4 and 8 weeks. BMPS were fixed in4% paraformaldehyde for 1 hour, washed 3 times in PBS, then incubatedfor 1 hour in blocking solution consisting of 5% normal goat serum (NGS)in PBS with 0.4% TritonX (Sigma). BMPS were then incubated at 4° C. for48 hours with a combination of primary antibodies (Table 2) diluted inPBS containing 3% NGS and 0.1% TritonX. BMPS were washed in PBS 3 timesafter which they were incubated with the appropriate fluorophore-taggedsecondary antibody for 1 hour in PBS with 3% NGS at room temperature.Double immunostaining was visualized using the proper combination ofsecondary antibodies (e.g., goat anti-rabbit secondary antibodyconjugated with Alexa 594 and goat anti-mouse secondary antibodyconjugated with Alexa 488 (Molecular Probes). Nuclei were counterstainedwith DRAQS dye (Cell Signaling; 1:5000 in 1×PBS) or NucRed Live(Molecular Probes) for 15 minutes before mounted on slides withcoverslips and Prolong Gold antifade reagent (Molecular Probes); BMPSused as negative controls for immunostaining were processed omitting theprimary antibody. Images were taken using a Zeiss UV-LSM 510 confocalmicroscope. The experiments were performed in duplicates; at least threedifferent fields of view were analyzed for each combination ofantibodies. 3D reconstruction was done using Imaris 7.6.4 software forscientific imaging.

TABLE 2 Primary Antibodies. Antibody Host Type Source Dilution NF-HRabbit Polyclonal Enzo  1:1000 GFAP Rabbit Polyclonal Dako 1:500 Olig 1Mouse Monoclonal Millipore 1:500 CNPase Mouse Monoclonal Millipore 1:500Calbindin Mouse Monoclonal SIGMA 1:500 NOGO-A Rabbit Polyclonal SantaCruz 1:500 Map2 Mouse Monoclonal Chemicon  1:1000 MBP/SMI99 MouseMonoclonal COVANCE  1:1000 SMI-32 Mouse Monoclonal Stenberger  1:2000Monoclonals Synaptophysin Mouse Monoclonal SIGMA 1:500 VGLUT1 RabbitPolyclonal Alpha Diagnostic 1:500 TH Mouse Monoclonal Millipore 1:250Nestin Rabbit Polyclonal Millipore 1:200 Ki67 Rabbit Polyclonal abeam1:100 Caspase3 Rabbit Polyclonal R&D 0.2 μg/ml OLIG1 Mouse MonoclonalMillipore 1:200 TUJ1 Mouse Monoclonal Stemcell 1:200 technologies S100BRabbit Polyclonal Santa Cruz 1:200

Automated Quantitation of Cell Types

BMPS was differentiated for 8 weeks. Randomly selected pictures fromthree experiments were acquired by confocal imaging and then analyzedwith a custom algorithm created with the Cellomics TargetActivation(Thermo Fisher Scientific, Pittsburgh, Pa.) image-analysis softwarepackage. With this algorithm, cells were identified based on DRAQSstained nucleus and quantified oligodendrocytes and astrocytes based onstaining of CNPase, NOGO1 and GFAP.

Myelination Assessment and Quantification

To calculate the percentage of axonal myelination, a semi-automatedcomputer platform was used, termed computer-assisted evaluation ofmyelin formation (CEM) [82], which uses NIH Image J built-in tools aswell as a Math lab processing functions. The results were generated aspixel counts and percent values. The percent of myelinated axons wascalculated by dividing the pixel count for myelin by the pixel count foraxons after cell body removal and multiplying by 100. For each timepoint at least 18 fields from at least two independent experiments wereanalyzed.

Electron Microscopy

BMPS aggregates were collected at 2, 4 and 8 weeks and were fixed in 2%glutaraldehyde and 4% formaldehyde in 0.1M Sodium Cacodylate buffer(EMS, electron microscopy sciences) pH 7.4 with 3% sucrose and 3 mMCaCl₂). Post-fixation was done with 2% osmium for 2 hours. The BMPSaggregates were then stained en bloc with 2% uranyl acetate in distilledwater for 30 min and subsequently dehydrated in graded ethanol. Embed812 (EMS) was used as the embedding media. Thin sections (70-80 nm) werecut on a Reichert Jung Ultracut E microtome and placed on formvar coated100 mesh copper grids. The grids were stained with uranyl acetate andfollowed by lead citrate. All imaging was performed on a Zeiss Libra 120electron microscope with a Veleta (Olympus) camera.

Treatment and Cytotoxicity Assay

BMPS was exposed to different concentrations of rotenone and MPP+ for 24and 48 hours after 4 weeks of differentiation. Rotenone workingsolutions were prepared in differentiation medium from 10 nM or 100 μMstocks to reach final concentrations of 0.1, 1, 10, 25 and 50 μM. DMSOwas used as vehicle control. MPP+ working solutions were prepared indifferentiation medium from 30 mM stocks to reach final concentrationsof 10, 50, 100, 500, 1,000, 5,000 and 10,000 μM. Four independentexperiments in 3 replicates were performed for each experimentalcondition (control and toxicant exposure for the different time points).Resazurin reduction assay was performed in order to determine cellviability after rotenone and MPP+ treatment. Resazurin(7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue dye that is reducedinto red fluorescent resorufin by redox reactions in viable cells. 100μl Resazurin (2 mg/ml stock) were added directly to the 6 well plates (2ml/well). Plates were incubated for 3 h at 37° C., 5% CO₂. Subsequently,50 μl of medium were transferred from each well in triplicates to a96-well plate and fluorescence was measured at 530 nm/590 nm(excitation/emission) using a multi-well fluorometric reader CytoFluorseries 4000 (PerSeptive Biosystems, Inc). Data were presented asmean±SD. Statistical analysis was performed using Dunnett's test.

Reactive Oxygen Species Measurement

Reactive oxygen species (ROS) were measured in cell media collected 24hours after treatment with 5 μM rotenone or 1,000 μM MPP+ using theOxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs, San Diego, Calif.).This is a fluorescence-based assay measuring the presence of total freeradicals within a sample and was used according to the manufacturer'sprotocol. The quenched fluorogenic dye dichlorodihydrofluorescin-DiOxyQ(DCFH-DiOxyQ) which is similar to the popular2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) is first primedwith a quench removal reagent. The resulted highly reactivenon-fluorescent DCFH can react with present ROS species in the cellsupernatant and is then oxidized to the highly fluorescent DCF(2′,7′-dichlorodihydroxyfluorescein). At every time point, 50 μl of thecell supernatant was added to a 96-well plate in triplicates and wasmixed and incubated with the DCFH-DiOxyQ for 45 minutes. Thefluorescence intensity was measured with a fluorescence microplatereader at 480 nm/530 nm (excitation/emission) and was proportional tothe total ROS/RNS levels within the sample.

Flow Cytometry

In order to quantify percentage of NPCs, and neurons within theaggregates, flow cytometry with NPC and neuronal markers was performed.Flow cytometry was performed according to previously published protocol[77] with some optimization steps for 3D cultures. Aggregates werewashed once with PBS/1 mM EDTA and trypsinized directly in the wellusing TrypLE Express containing 4 units/ml DNAse for 30 min at 37° C. onthe shaker. Pipetting the aggregates up and down with a 1 ml syringe anda 26G3/8 needle ensured generation of single cell suspension. Cells werecounted, washed once with PBS/1 mM EDTA, fixed with 2% PFA for 20 min at4° C., washed twice with PBS/1% BSA (wash solution I, WS I) and blockedfor 30 min in blocking solution (PBS/1% BSA/0.15% saponin/10% NGS).1×10⁶ cells were stained for one hour at 4° C. withfluorochrome-conjugated antibodies dissolved in blocking solution (Table3). Unstained cells as well as cells incubated with isotype controlswere used as negative controls to set the gates for measurements. Cellswere washed twice with PBS/1% BSA/0.15% saponin, once with PBS/1% BSA.Flow cytometry was performed using a Becton Dickinson FACSCalibur systemby measuring 10⁴ gating events per measurement. Data was analyzed usingFlowJo v10 software.

TABLE 3 Antibodies for flow cytometry analysis Antibodies Host typeSource Dilution Alexa Fluor ® Mouse Monoclonal, BD Pharmingen 1:05 647Nestin clone 25 Alexa Fluor ® Mouse Monoclonal, BD Pharmingen 1:05 488β-III- clone TUJ1 Tubulin PerCP-Cy ™ Mouse Monoclonal, BD Pharmingen1:20 5.5 Sox2 clone 030-678 PerCP-Cy ™ Mouse Monoclonal, BD Pharmingen1:20 5.5 Sox1 clone N23-844 PE Doublecortin Mouse Monoclonal, BDPharmingen 1:20 clone 30 Alexa Fluor ® Mouse Monoclonal, BD Pharmingen1:20 647 Ki67 clone B56

Microelectrode Array (MEA) Recordings

After 8 weeks of differentiation, BMPS were plated on 48-well MEA platespreviously coated with Matrigel. During two weeks spontaneous electricalactivity was recorded using the ‘Maestro’ MEA platform and Axion'sIntegraded Studio (AXIS) software [Axion Biosystems inc.; Atlanta, US].Each well of the 48-well MEA plate contains 16 individualmicroelectrodes (˜40-50 μm diameter, center-to-center spacing 350 μm)with integrated ground electrodes, resulting in a total of 768electrodes/plate. The ‘Maestro’ MEA platform has an integrated heatingsystem, which can be controlled by AXIS software. All recordings wereperformed at a constant temperature of 37° C. Prior to a twenty minutesrecording, the MEA plates were placed in the Maestro MEA platform andequilibrated for five min. AXIS software was used to control heatingsystem and monitor the recordings, which includes simultaneouslysampling of the channels at 12.5 kHz/channel with a gain of 1200× and aband pass filter of 200-5000 Hz. The recordings were converted into RAWfiles. After a recording the RAW-files were re-recorded with AXIS toconvert the data into a spike file, which includes spike timing andprofile information. A variable threshold spike detector was used forthe spike-file, it was set at 6 times standard deviations of therms-noise on each channel. The spike file was later used for dataanalysis with NeuroExplorer® [Nex Technologies, Madison (AL), US] toconvert data into Microsoft Excel files. Using the function ratehistogram, a summary of the spikes of all electrodes of one plate wasput into one Excel sheet. Only electrodes that recorded activity higherthan 0.05 spikes/sec at least once over the time measured were includedfor data analysis.

Statistical Analysis

Statistical analysis was performed using GraphPad InStat 3. TheDunnett's test was applied to all the experiments shown here thatcompare to a control group. Statistically significant values (p<0.01)are marked with an asterisk (*). For myelination quantification at thedifferent time points, a Kruskal-Wallis test was employed, statisticalsignificance was considered for p values <0.05.

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Example 14

Human induced pluripotent stem cells (iPSCs), together with 21st centurycell culture methods, have the potential to better model humanphysiology with applications in toxicology, disease modeling, and thestudy of host-pathogen interactions. Several models of the human brainhave been developed recently, demonstrating cell-cell interactions ofmultiple cell types with physiologically relevant 3D structures. Mostcurrent models, however, lack the ability to represent the inflammatoryresponse in the brain because they do not include a microglial cellpopulation. Microglia, the resident immunocompetent phagocytes in thecentral nervous system (CNS), are not only important in the inflammatoryresponse and pathogenesis; they also function in normal braindevelopment, strengthen neuronal connections through synaptic pruning,and are involved in oligodendrocyte and neuronal survival. Here, we havesuccessfully introduced a population of human microglia into 3D humaniPSC-derived brain spheres (BrainSpheres, BS) through co-culturing cellsof the Immortalized Human Microglia—SV40 cell line with the BS model(OS). We detected an inflammatory response to lipopolysaccharides (LPS)and flavivirus infection, which was only elicited in the model whenmicroglial cells were present. A concentration of 20 ng/mL of LPSincreased gene expression of the inflammatory cytokines interleukin-6(IL-6), IL-10, and IL-1b, with maximum expression at 6-12 hpost-exposure. Increased expression of the IL-6, IL-1b, tumor necrosisfactor alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) geneswas observed in μBS following infection with Zika and Dengue Virus,indicating a stronger inflammatory response in the model when microgliawere present than when only astrocyte, oligodendrocyte, and neuronalpopulations were represented. Microglia innately develop within cerebralorganoids (Nature communications)′, the findings indicate that the μBSmodel is more physiologically relevant and has potential applications ininfectious disease and host-pathogen interactions research.

INTRODUCTION

Microphysiological systems and organotypic cell culture models areincreasingly being used to model human physiology in vitro because thesemodels better mimic the in vivo situation compared to traditionalmonolayer cell culture systems (Marx et al., 2016; Pamies and Hartung,2017; Smirnova et al., 2018). In addition, such models allow for themechanistic understanding of biological processes that would bechallenging to study in vivo. In particular, the human brain is a highlycomplex structure that cannot easily be interrogated in vivo and hasproven difficult to model in vitro. Recently, the first in vitro humanorganotypic models of the brain have been developed, including 3Dstructures and heterogeneous cell populations. Many of these models seekto represent key events in human brain development and cell-cellinteractions between various cell types (Kadoshima et al., 2013;Lancaster et al., 2013; Pasca et al., 2015; Pamies et al., 2017;Sandstrom et al., 2017). The basis for the majority of these models is aneural progenitor cell (NPC) population; however, microglia, arise fromerythromyeloid precursor cells in the embryonic yolk sac, unlike theother cell types in the brain which arise from the neuroectoderm (Alliotet al., 1999; Ginhoux et al., 2013; Kierdorf et al., 2013; Sousa et al.,2017). Thus, because microglia cannot be derived from NPCs, many of thecurrent in vitro models of the human brain do not include a microglialcell population.

Microglia are the resident mononuclear phagocytic cells in the centralnervous system (CNS) (Ginhoux et al., 2013). These cells are bothneuroprotective and immunocompetent, as they play critical roles inbrain development, strengthen neuronal connections through synapticpruning, are involved in neuronal maintenance and support, and areresponsible for the inflammatory response in the brain following aninjury or pathogenic infection (Kettenmann et al., 2011; Paolicelli etal., 2011; Michell-Robinson et al., 2015; Thompson and Tsirka, 2017). Inresponse to local alterations, lesions, or pathogen invasion, microgliabecome active. Microglial activation involves changes in morphology, therelease of multiple substances such as proinflammatory cytokines,chemokines, and reactive oxygen species, migration to affected areas,proliferation, and phagocytosis of cell debris (Kettenmann et al., 2011;Boche et al., 2013; Thompson and Tsirka, 2017). This process is also aprominent feature of inflammation in neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, multiple sclerosis, andinfectious processes (Wang et al., 2015; Thompson and Tsirka, 2017).Various models have been developed to study neurological disorders andlipopolysaccharide (LPS) has been widely used as an inducer ofneuroinflammation and neurotoxicity as it has been shown to act as apotent stimulator of microglia (Gao et al., 2002; Hu et al., 2012;Olajide et al., 2013; Kempuraj et al., 2017). Additionally, inflammationrelated neurodegeneration induced by LPS is a common approach to studymechanisms of cellular neuroimmunology (Hu et al., 2012). Neurotropicvirus infections are major pathogens in the CNS and cause inflammation(Ludlow et al., 2016). The Flavivirus genus constitutes some of the mostserious human pathogens, including Japanese encephalitis (JEV), dengue(DENV), zika (ZIKV), and yellow fever (YF), all of which are can invadethe central and peripheral nervous system and are regarded asneurotropic viruses (Neal, 2014). The mechanisms by which flavivirusesalter the immune and the CNS have only recently been examined, butremain largely unclear (Daep et al., 2014). The NPC model has been usedin ZIKV studies to understand mechanisms that govern neuropathogenesisand immunopathogenesis in CNS infection (Dang et al., 2016; Garcez etal., 2016; Tang et al., 2016; Qian et al., 2017), but these models donot contain microglia. We previously developed a 3D brain model(BrainSpheres, BS) derived from human-induced pluripotent stem cells(iPSCs) and demonstrated that this model is reproducible with respect tosize and shape and is comprised of populations of neurons, astrocytes,and oligodendrocytes (Pamies et al., 2017). In addition, the model hasbeen applied to the study of developmental neurotoxicity (Pamies et al.,2018). In this study, we introduced a population of human microglia intothe BS model and compared the inflammatory response of BS withoutmicroglia to BS co-cultured with microglia (μBS) after treatment withLPS, ZIKV, or DENY. In addition, we quantified cell death following ZIKVinfection because there is evidence that ZIKV infection can impair braingrowth via cell death of developing neurons.

Materials and Methods

Induced Pluripotent Stem Cell Generation

Fibroblasts (American Type Culture Collection (ATCC), Manassas, Va.,United States, CCD-1079Sk ATCC® CRL-2097™) were used to generate iPSCsas previously described (Wen et al., 2014; Pamies et al., 2017). iPSCs(passage ≤20) were cultured on irradiated MEFs in human embryonic stemcell (hESC) medium comprised of D-MEM/F12 (Invitrogen, Carlsbad, Calif.,United States) 20% Knockout Serum Replacement (KSR, Invitrogen,Carlsbad, Calif., United States), 2 mM L-glutamine (Invitrogen,Carlsbad, Calif., United States), 100 μM MEM NEAA (Invitrogen, Carlsbad,Calif., United States), 100 μM β-mercaptoethanol (Invitrogen, Carlsbad,Calif., United States), and 10 ng/mL human basic FGF (bFGF, PeproTech,Rocky Hill, N.J., United States). Cell culture medium was changed daily.iPSCs were passaged using collagenase (Invitrogen, Carlsbad, Calif.,United States, 1 mg/mL in D-MEM/F12 for 1 h at 37° C.).

Neural Progenitor Cell Production

NPC were generated following the previously published protocol (Wen etal., 2014). NPCs were cultured with StemPro® NSC SFM media (LifeTechnologies, Carlsbad, Calif., United States) in 175 cm² flasks (ThermoScientific, Waltham, Mass., United States, Nunc™ Filter Cap EasYFlask™)coated with Poly-L-ornithine hydrobromide (Sigma-Aldrich, St. Louis,Mo., United States, P3655) and laminin (Sigma-Aldrich, L2020, fromEngelbreth-Holm-Swarm murine sarcoma basement membrane). Half of themedia was changed daily. Cultures were maintained at 37° C. with 5% CO2.NPCs were passaged by mechanical detachment with a cell scraper(Sarstedt, N.C., United States, 2-position, Blade 25,83.1830).

BrainSpheres Differentiation

The production of BS was published previously (Pamies et al., 2017).Briefly, to produce BS, NPCs were detached mechanically with a cellscraper (Sarstedt, 2-position, Blade 25, 83.1830), re-pipetted fordisaggregation, and counted using the Countess Automated Cell Counter(Invitrogen, Carlsbad, Calif., United States). 2×10⁶ cells per well wereplated in non-treated Falcon™ Polystyrene 6-well plates (Corning,Corning, N.Y., United States). Cells were grown in differentiationmedium [Neurobasal® electro Medium (Gibco, Gaithersburg, Md., UnitedStates)] supplemented with 2% B-27® Electrophysiology (Gibco), 1%GlutaMAX (Gibco), 0.01 μg/mL human recombinant GDNF (Gemini, Woodland,Calif., United States), and 0.01 μg/mL human recombinant BDNF (Gemini).Cultures were maintained at 37° C. in an atmosphere of 5% CO2 underconstant gyratory shaking (88 rpm) for 7 weeks. Differentiation mediumwas changed every 2 days.

Microglia Culture

Immortalized Human Microglia—SV40 (Applied Biological Materials Inc.,Richmond, BC, Canada) were grown in non-treated 75 cm² flasks (ThermoScientific, Waltham, Mass., United States, Nunc™ Filter Cap EasYFlask™).Cells were expanded using Prigrow III medium (Applied BiologicalMaterials Inc.) supplemented with 10% Fetal Bovine Serum (Gibco,Gaithersburg, Md., United States, Certified, Heat Inactivated, UnitedStates Origin) and 1% Penicillin-Streptomycin (Gibco, 10,000 U/mL).Microglia were passaged by mechanical detachment using a cell scraper(Sarstedt, 2-position, Blade 25, 83.1830). Cultures were maintained at37° C. in an atmosphere of 5% CO₂ immortalized Human Microglia—SV40 cellline was declared mycoplasma free by a mycoplasma testing using a PCRbased MycoDtect kit from Greiner Bio-One (Genetic Resource Core Facilityis a core resource of the Johns Hopkins School of Medicine, Institute ofGenetic Medicine).

Formation of BS with Microglia (pBS)

The density of microglia added to the aggregates was studied previouslyand the optimal number of cells was chosen to avoid the formation ofmicroglia-only spheres and to allow enough microglia attachment toaggregates. BS were differentiated for 7 weeks at which time, 3×10⁵microglia/well were added to the BS suspension. Upon addition ofmicroglia, plates were kept in static conditions in the incubator at 37°C. with 5% CO2 for 24 h, with manual shaking of the plates every 6 h.This allowed for the attachment of the microglia to the surface of theBS while avoiding BS agglomeration. Microglia which did not attach to BSduring this time attached to the bottom of the well. We then carefullyremoved aggregates from the well and transferred them to a new plate.μBS aggregates were then washed twice with PBS, and new differentiationmedia was added. After the incorporation of microglia, μBS weremaintained in culture for one more week.

Histology and Immunohistochemistry

BS with and without incorporated microglia were fixed in 4%formalin/PBS. After fixation, spheres were carefully embedded intolow-melting agarose and the solidified mixture was processed intoparaffin blocks using an ASP300S dehydration machine (Leica, Wetzlar,Germany) and an EG1160 tissue embedding system (Leica, Wetzlar, Germany)Sections (2 μm) were stained with hematoxylin-eosin (H&E) or processedfor immunohistochemistry as follows: After dewaxing and inactivation ofendogenous peroxidases (PBS/3% hydrogen peroxide), antibody specificantigen retrieval was performed using the Ventana Benchmark XT machine(Ventana, Tucson, Ariz., United States). Sections were blocked (PBS/10%FCS) and afterward incubated with the primary antibodies TMEM119 (1:100;Sigma), Mertk (1:100; R&D), Ax1 (1:100; LSBio), CD11b (1:2,000; Abcam),and P2ry12 (1:100; Sigma). For double staining of microglia and neurons,microglia were detected with TMEM119 and visualized with DAB in brown,whereas neurons were stained with NeuN (1:50; Millipore) and incubatedwith anti-mouse AP-coupled secondary antibody and developed in red.Bound primary antibodies were detected with secondary antibodies usingHistofine Simple Stain MAX PO immune-enzyme polymer (NichireiBiosciences) and stained with 3,3′-Diaminobenzidine (DAB) substrateusing the ultraView Universal DAB Detection Kit (Ventana). Tissues werecounterstained with hematoxylin. Representative images were taken with aLeica DMD108 digital microscope.

Virus Propagation and Titering

Vero (ATCC, Manassas, Va., United States, ATCC® CCL81™) and BHK-21(ATCC®, C-13 ATCC CCL-10) cell lines were cultured in D-MEM (Gibco)containing 10% Fetal Bovine Serum (Gibco, Certified, Heat Inactivated,United States Origin), 2 mM L-glutamine (Invitrogen),Penicillin-Streptomycin (Gibco 10,000 U/mL), and 10 mM Hepes buffer(Gibco) in an incubator at 37° C. with 5% CO₂. Vero and BHK-21 cellswere passaged 3 times a week. To passage Vero and BHK-21 cells, thecells were incubated with 0.05% trypsin (Gibco) in 1×PBS (Gibco) at 37°C. with 5% CO₂ for 5 min, resuspended in 10 mL D-MEM, and centrifuged at1200 rpm for 8 min. The supernatant is then removed and the cells wereresuspended in fresh media and transferred to a new flask.

Two strains of ZIKV (ATCC, VR-1838™, Uganda 1947, ZIKV-UG, and Brazil2015, ZIKV-BR) were propagated in Vero cells and Dengue Virus type 1(ATCC, VR-1586™, DENV-1) was propagated in BHK-21 cells. ZIKV-BR wasisolated from a febrile non-pregnant Brazilian woman with a rash inParaiba, Brazil in 2015 and kindly provided by Professor PedroVasconcelos from the Instituto Evandro Chagas, Belem, State of Para,Brazil (Pompon et al., 2017). ZIKV-UG was isolated in 1947 from a rhesusmacaque exposed to mosquitos in Uganda. The Viral stocks were preparedby infecting Vero or BHK-21 cells at 80-90% confluency in 25 cm² flaskswith 2 mL of virus dilution in OptiPro™ SFM media (Gibco) at amultiplicity of 0.1. The cells were incubated for 6 h at 37° C. with 5%CO2, then the supernatant was removed and the cell lines were washed twotimes with 1×PBS and replaced with 10 mL of D-MEM. The infected cellswere further incubated for 5 days, and then the supernatants werecollected and transferred to a 75 cm² flask with respective cell linesat 90% confluence. The ZIKV-UG, ZIKVBR, and DENV-1 were collected after7 days post-infection (p.i.), clarified by centrifugation at 500×g for10 min, and filtered through a 0.22-μm membrane. All viral stocks werestored in 1 mL aliquots at −80° C. Virus titers used in the assays weredetermined by double-overlay plaque assay of Vero and BHK-21 cells toZIKV and DENV, respectively, as previously described (Baer andKehn-Hall, 2014).

Lipopolysaccharide Treatment

After 8 weeks of differentiation, 2 μl from a 10 μg/mL stock of LPS wereadded to 2 mL media containing ether the microglia, BS or μBS, obtaininga final concentration of 20 ng/mL. Samples were collected 3, 6, 12, and24 h after the exposure. Time 0 refers to samples before LPS exposure.Samples were collected for qPCRs.

Viral Infection

Immortalized Human Microglia—SV40 were plated in 24-well plates at adensity of 2.5×10⁵ cells per well. The BS and μBS or microglia from onewell were transferred to a 24-well-plate (and divided into triplicates)before infecting with ZIKV-UG, ZIKVBR, and DENV-1. A MOI of 0.1 for 6 hat 37° C. with 5% CO₂ was used. Three wells of non-infected BS, μBS, andmicroglia were used as control (MOCK). The cells were washed 3 timeswith 500 μl PBS, and then 250 μl of supernatant were collected for eachcondition as time zero of infection. The PBS was totally removed and BS,μBS or microglia were re-suspended in fresh complete media. BS and μBSwere then transferred back to a 6-well plate containing media. Theplates containing the infected and uninfected cells were incubated at37° C. with 5% CO₂ for 24 to 72 h. The supernatant was collected atdifferent times points 24, 48, and 72 h post infection (p.i.) to checkviral load. The cells were collected at 48 and 72 h p.i. to analyze geneexpression, Annexin-V, and cell cycle (3 technical replicates). One wellof BS, μBS and microglia were used for confocal microscope analysis. Allwork with infectious ZIKV was performed in an approved BSL-3 facility.

Analysis of Cell Viability and Cytotoxicity Using Flow Cytometry

Apoptosis in microglia cells, BS, and μBS was assayed using the MuseAnnexin V and Dead Cell kit (Millipore, Billerica, Mass., United States)according to the user guide and the manufacturer's instructions. After72 h incubation at 37° C. in 5% CO₂, microglia cells for all conditionswere harvested through mechanical detachment with a cell scraper(Sarstedt, Blade 25, 83.1830) in 500 μl of the PBS, and centrifuged for5 min at 2000 RPM. Two aggregates of BS and μBS were collected using a 1mL micropipette. The pellet was resuspended in 100 μL of the PBS with 1%FBS, mixed with 100 μL of the Muse Annexin V and Dead Cell Reagent atroom temperature. Tubes were mixed using a vortex for 10 s. Samples wereincubated for 20 min at room temperature and protected from light. Thepercentages of apoptotic cells were analyzed by flow cytometry usingMuse Cell Analyzer (Millipore, United States) system and values wereexpressed as mean of apoptotic cells relative to mock with error barsrepresenting the SD. All values are expressed as mean±SD (n=3).Statistical significance (defined as P-value <0.05) was evaluated usingmultiple t-test to compare control and infected or treated cells (GraphPrism 7 Software, San Diego, Calif., United States).

Flow Cytometry Analysis of Cell Cycle

Microglia cells, BS and μBS (infected and uninfected) were cultivated in6-well plates and incubated for 48 h. Microglia cells for all conditionswere harvested through mechanical detachment with a cell scraper(Sarstedt, Blade 25, 83.1830) in 500 μl of the PBS, and centrifuged for5 min at 2000 RPM. Two aggregates of BS and μBS were collected using a 1mL micropipette. Obtained pellets were fixed with 70% ethanol. The cellswere kept in −20° C. overnight. After ethanol removal, cells weresuspended in 250 μL PBS and centrifuged for 5 min at 2000 RPM. Cellpellets were suspended in 200 μL of Muse Cell Cycle Reagent and wereincubated for 30 min at room temperature and protected from light. Thecell suspension was transferred to a 1.5 mL microcentrifuge tube priorto analysis on Muse Cell Analyzer. Cell cycle was assessed byfluorescence-activated cell analysis using a Muse Cell Analyzer (Merck,Millipore, United States). All values are expressed as mean±SEM (n=3).Statistical significance (defined as P-value <0.05) was evaluated usingmultiple t-test to compare control and infected or treated cells withLPS (Graph Prism 7 Software).

Viral RNA Extraction

Viral RNA for the real-time RT-PCR (qPCR) assays was purified from theculture supernatant of the uninfected and infected microglia, BS andμBS. The viral RNA was extracted from 240 μl of culture supernatant byusing the QlAamp MinElute virus spin kit (Qiagen, Valencia, Calif.,United States) according to the manufacturer's instructions, with theexception of the elution volume, which was 65 μl.

DENV-1 and ZIKV qPCR

RNA isolated from the supernatant after DENV-1 and ZIKV infections wereused in qPCR independently. The MOCK samples were used as negativecontrols in both qPCRs. The QuantiTect One-Step RT-PCR kit (Qiagen,Hilden, Germany) was used with a 25 μl reaction mixture under thefollowing conditions: 5 μl of kit master mixture (including Taqpolymerase and RT enzyme), 1.25 μl of 10 μM of each primer, 0.5 μl of 10μM of probe, 7 μl of RNA-free-water (Mol Bio grade, Hamburg, Germany),and 10 μl of the extracted sample. Each amplification run containednegative control (NC) of each experiment (MOCK); non-template control(NTC), and positive control (PC). The non-template control consisted ofblank reagent and water. For the positive control, nucleic acidextracted from each virus stock were used after dilution 1:1000 to avoidcross-contamination. The protocol used to ZIKV and DENV-1 qPCR was thesame conditions except primers and probes (Table 4) and cycling thermal.The DENV-1 qPCR was done as described previously (Johnson et al., 2005)with some modification; a single cycle of reverse transcription for 30min at 50° C., 15 min at 95° C. for reverse transcriptase (RT)inactivation, and DNA polymerase activation followed by 45 amplificationcycles of 15 s at 95° C. and 1 min 55° C. (annealing-extension step).The following thermal profile was used to ZIKV qPCR a single cycle ofreverse transcription for 30 min at 50° C., 15 min at 95° C. for RTinactivation, and DNA polymerase activation followed by 40 amplificationcycles of 15 s at 95° C. and 1 min 60° C. (annealing-extension step).For RNA standards, RNA was isolated from purified, titered stock ofZIKV-UG or DENV-1. RNA yield was quantified by spectrometry and the datawas used to calculate genomes/μl. ZIKV-UG and DENY-1 RNA was seriallydiluted 1:10 in 8 points of dilution for standard curve (10⁷ to 10¹).The standard curve was amplified in duplicate using primers andconditions described above for each specific qPCR. The number ofinfectious viral RNA transcripts detected was calculated by generating astandard curve from 10-fold dilutions of RNA isolated.

TABLE 4 Oligonucleotide primers and fluorogenic probesused in DENV-1 and ZIKV virus qPCR assay. Virus Genome posi- Fluoro-detection Sequence 5′-3′ tion phone DENV-1  CAAAAGGAAGTCGTGCAATA 8978- —reverse  8993 primer DENV-1  CTGAGTGAATTCTCTCTACTGAACC 9084- — reverse 9109 primer DENV-1  CATGTGGTTGGGAGCACGC 8998- FAM/ probe 9017 BHQ-1ZIKV  AARTACACATACCARAACAAAGTGGT 9271- — forward  9297 primer ZIKV TCCRCTCC CYCTYTGGTCGGT 9352- — reverse  9373 primer ZIKV CTYAGACCAGCTGAAR 9304- FAM/ probe 9329 BHQ-1

Analysis of Confocal Microscopy

After 72 h exposure BS and μBS were collected and fixed with PFA (4%)for 1 h at room temperature. Subsequently, BS, and μBS were washed twicewith PBS (1×) and incubated with blocking solution (10% goat serum, 1%BSA, 0.15% saponin in PBS) at 4° C. for 1 h. The BS were washed withwashing solution (1% BSA, 0.15% saponin in PBS), and incubated overnightat 4° C. with primary antibodies: Zika NS1 (OWL, 55788, 1:200, ZIKV)anti-NF200 (Sigma Aldrich, N4142, 1:200, neurofilament for all neurons),and IBA1 (WAKO, Richmond, Va., United States, 019-19741, 1:200,microglia) diluted in blocking solution. The next day the BS and μBSwere washed twice with washing solution and incubated with the secondaryantibody 568-Alexa anti-rabbit and 488-Alexa anti-mouse both 1:200diluted in blocking solution for 24 h at 4° C. and protected from light.Thereafter, BS and μBS were washed twice again with washing solution.Nuclei were stained with Hoechst 33342, diluted 1:10,000 in PBS for 1 h.The BS were transferred to microscope glass slides and mounted with“Immuno mount” and imaged with a confocal microscope Zeiss LSM 510Confocal III (Zeiss) with a 20× objective.

Cellular RNA Isolation and qPCR

Total RNA was extracted from microglia, BS and μBS 72 h post infection(p.i.) using RNAeasy Mini Kit (Qiagen, Hilden, Germany). RNA quantityand purity was determined using a NanoDrop 2000c (Thermo Scientific). 1μg of RNA was reverse-transcribed using the MLV Promega RT (Promega)according to the manufacturer's recommendations. The expression of geneswas evaluated using specific TaqMan® Gene Expression Assays (LifeTechnologies). Table 5 shows a summary of the assayed genes. qPCR wasperformed using a 7500 Fast Real Time system machine (AppliedBiosystems). Fold changes were calculated using the 2 (−ΔΔCt) method(Livak and Schmittgen, 2001). β-actin and 18s were used as housekeepinggenes for mRNA. Data are presented as mean±SD, normalized tohousekeeping genes and MOCK.

TABLE 5 List of TaqMan assays used for QPCR analysis CCL2 Chemokine (C-Cmotif) ligand 2 Hs00234140_m1 TNF-α Tumor necrosis factor alphaHs01113824_g1 IL10 Interleukin 10 Hs00961622_m1 IL6 Interleukin 6Hs009S5839_m1 IL1b Interleukin 1 beta Hs01555410_m1 ACTB Beta-actinHs01060665_g1

Results

Incorporation of SV40-Immortalized Human Microglial Cells Into a 3D BSModel

Microglia cells were added to the BS at 7 weeks of differentiation (FIG.9A) and were able to attach to the surface of the BS. However, as themicroglia cell line used for this study

is immortalized and proliferation takes place, we were only able tomaintain (IBS (BS with microglia) for 1 week after incorporation for ourexperiments Immunohistochemistry of (IBS demonstrated they were positivefor microglia markers such as TMEM19, Mertk, Ax1 (FIG. 9B), and IBA1(FIG. 9C). Microglia grew on the surface of the μBS, forming a microgliaprotuberance on the sphere (FIGS. 9C,D). These formations appear in 50%of the aggregates, seemingly due to the co-culture technique.

Presence of Microglia in BS Alters Gene Expression of Cytokines inResponse to Inflammatory Stimuli

Once microglial cells were successfully incorporated into BS, weevaluated whether the μIBS model would then respond to inflammatorystimuli in a distinct manner to BS. Microglial culture, BS, and μIBSwere exposed to 20 ng/mL LPS, which is a potent activator ofinflammatory pathways in myeloid cells (Fujihara et al., 2003). Cellswere collected at 0, 3, 6, 12, and 24 h post-treatment (p.t.) and levelsof CCL2, TNF-α, IL-1b, IL-6, and IL-10 RNA were quantitated by qPCR. Inthe absence of microglia, BS responded poorly to LPS (FIG. 10A).Conversely, cultures containing solely microglial cells responded to LPSby upregulating the expression of CCL2 and TNF-α 24 h p.t. Nosignificant changes in the levels of IL-6, IL-1b, and IL-10 wereobserved. The mixing of both populations into uBSs, however, led todistinct responses against LPS, specifically for IL-1b, IL-6, and IL-10,which were all upregulated in the first 12 h p.t. and downregulated 24 hp.t. The increased RNA expression of CCL2 and TNF-α in sole microgliacultures was not observed in μBS cultures, indicating that the presenceof other cell types in the BS indirectly (through secreted factors) ordirectly (by cell-to-cell interactions) altered the LPS-response, whichwas observed in myeloid cells. This indicates crosstalk betweenmicroglia and other cell populations in the μBS model. After LPStreatment, microglia showed an increase in dividing cells, characterizedby a higher percentage of cells in S or G2/M phases when compared tonon-treated microglia (FIG. 10B). In addition, cell viability decreasedafter LPS treatment in microglia and in μBS, indicating that microgliamay play an important role in viral neurotoxicity (FIG. 10C).

The μBS Support Replication of Dengue 1 (DENV-1) and Zika Viruses (ZIKV)

Organoids have been previously used as models for virus pathogenesis(Gabriel et al., 2017; Salick et al., 2017; Watanabe et al., 2017). Toevaluate whether the incorporation of microglial cells into BS wouldinterfere with viral replication, microglia, BS, and μBS were infectedwith DENY-1 and two distinct strains of ZIKV (ZIKV-UG and ZIKV-BR). FIG.11 represents immunohistochemistry on BS and μBS for control (mock) andtreated samples (ZIKV-BR, ZIKV-UG, and DENY). Mock samples did not showany positive staining for ZIKV (FIG. 11) or DENY (data no shown).Cultured cells and aggregates were exposed to the three pathogens andviral growth in the supernatants was measured every 24 h for 3 days(FIG. 12A). Microglia and μBS showed higher replication efficiency inthe first 48 h showing a significant difference after 72 h in microgliainfected with ZIKV-UG and ZIKV-BR. However, DENY-1 did not showsignificant changes between models after 72 h (FIG. 12A). Viralinfection was confirmed by immune staining (FIG. 11). Both BS and μBSwere found to be infected by ZIKVBR, ZIKV-UG, and DENY which uponqualitatively analyzing the images, showed higher virus accumulation inmicroglia cells than in the other cell types (FIG. 11). This togetherwith the replication results (FIG. 12A) indicate that these flavivirusinfect microglia or that microglia internalize more viral particles viaphagocytosis. DENY-1 infection led to a slight increase in dividingcells in BS and μBS, characterized by a higher percentage of cells in Sor G2/M phases when compared to sole microglia (FIG. 12B). However,DENY-1 was slightly more cytotoxic to microglia and BS than μBS (FIG.12C), indicating a protective role for microglial cells in the 3D model.Similar results were observed against ZIKV-UG, which appeared to be morecytotoxic to BS (FIG. 12C), despite no changes in the cell cycle amongall conditions (FIG. 12B). This could be due to the protectivecapability of microglia and their role in internalizing viral particles.Interestingly, ZIKV-BR infection in μBS caused a significant increase individing cells concomitantly to a slight decrease in viability (notstatistically significant), indicating that microglial cells contributeto higher cytotoxicity in response to a more aggressive ZIKV strain.

The Presence of Microglia Alters the Cytokine Gene Expression inResponse to Flavivirus Infection

As part of the innate immune response, microglial cells respond to viralinfection by secreting inflammatory cytokines. To determine whether theaddition of microglial cells to BS modulates the immune response againstflavivirus infection, the levels of intracellular TNF-α, CCL2, IL-1b,and IL-6 mRNA were measured in sole microglia, BS, and μBS 72 h afterexposure to DENY-1, ZIKV-UG, and ZIKV-BR. After DENY-1 infection,cytokine expression was observed mostly in microglia and μBS, but notBS. Interestingly, the secretion pattern varied between the analytes(FIG. 12D). While TNF-α and IL-6 production increased in sole microgliapost-infection, CCL2 and IL-1b, were upregulated only in μBS (FIG. 12D).The presence of microglia cells in μBS also led to an increase inZIKV-induced cytokine production when compared to sole microglia and BS.Moreover, cytokine levels in μBS were significantly higher in responseto ZIKV-BR than those observed with the Uganda strain.

Discussion

3D brain models have been increasingly used in the last years, primarilyin ZIKV studies (Qian et al., 2016, 2017; Wells et al., 2016; Salick etal., 2017; Watanabe et al., 2017; Zhou et al., 2017). These studies,however, have mainly focused on neurogenesis. The relevance of otherbrain populations such as microglia has been highlighted, indicatingthat these tissue-resident brain macrophages are key players in brainhomeostasis, development, and diseases (Derecki et al., 2014; Salter andStevens, 2017). Microglia incorporation into 3D models has not beenachieved previously. In this study, we have successfully introducedmicroglia into our 3D brain spheroids, and demonstrated they alter theresponse to viral infection. Two techniques were compared to establishthe protocol: in the first approach, human microglia immortalized cellswere added on top of the NPCs before the formation of the spheres, andthen differentiated in 3D over 8 weeks (FIG. 13). Without wishing to bebound by theory, the nature of the microglia used (immortalized by SV40)made this initial protocol impossible due to the constant proliferationleading to a high microglia population. Immortalized cell lines tend tobe used as a high-throughput model for experimentation as they arehomogeneous in culture. The human microglia immortalized with SV-40 usedin our study exhibits biological characteristics such as morphological,phenotypical, and functional properties similar to those documented inprimary human microglia (Patel et al., 2016; Zhu et al., 2016; Sousa etal., 2017). Also, immortalized microglia demonstrate appropriatemigratory and phagocytic activity, and can elicit the pro-inflammatorycytokine response, which is characteristic of human microglia(Garcia-Mesa et al., 2017). When adding microglia cells to NPCs (FIG.13, cells added during the aggregation stage with the NPCs), around 50%of the spheres led to overgrowth of microglia and some neuronal celldeath. The use of iPSC-derived microglia, however, could solve thisproblem in the future (Muffat et al., 2016). In order to avoidmicroglial proliferation, microglia cells were added after BSdifferentiation in the second regimen, after 7 weeks differentiation,and kept for a maximum of 1 week in co-culture (FIG. 9A). Microglia werethen identified by using different markers such as TMEM119, Mertk, Ax1,and IBA1, showing their attachment and growth in the μBS (FIGS. 9B,C).The pro-inflammatory stimulus LPS from Gram-negative bacteria as usedhere and Gram-positive lipoteichoic acid as shown earlier (Kinsner etal., 2005, 2006; Boveri et al., 2006) activate microglia in infections.LPS was used to challenge the μBS system and to compare its effects withboth BS and microglia cultures. In response to LPS, microglia producelarge quantities of proinflammatory cytokines such as TNF-α, IL-1b, andIL-6. This is normally followed by the production of anti-inflammatorycytokine IL-10 (de WaalMalefyt et al., 1991; Randow et al., 1995).Microglia cultures showed no statistically significant changes neitherin pro-inflammatory marker RNA levels (IL-6 and IL-1(3) or in theanti-inflammatory marker (IL-10). This could be due to the need of otherCNS cell types such as astrocytes for the release of these specificcytokines. However, TNF-α and CCL2 were strongly upregulated 24 h afterLPS treatment (FIG. 10A) since microglia is the major producer of TNFαand CCL2 is a gene that encodes a small cytokine that recruits monocytesbetween other inflammatory cells (such as memory T cells and dendriticcells) into the inflammation area. Microglia exposed to LPS showedhigher expression (lower Ct values) of most of the genes studiedcompared to μBS and BS (data no shown). This indicates that microgliaexpress higher levels of these genes, or that in the presence of othercell types in the μBS, these dilute the expression levels of these genesas other cells do not express them (but expression is normalized tohousekeeping genes expressed by all cell types). Pro-inflammatorycytokine-encoding genes IL-6 and IL-1β showed a clear increase inexpression (FIG. 10A) in μBS. Interestingly, the anti-inflammatorymarker IL-10 was also upregulated after LPS treatment in μBS. Cytokinesare released very rapidly after inflammatory stimuli and are regulatedwithin a few hours, this could explain the observed co-expression ofboth anti- and pro-inflammatory genes. The TNF-α gene was not modifiedsignificantly after LPS exposure in μBS, BS, and microglia at the earlytime points. However, microglia exposed to LPS during 24 h LPS exposureshowed a high up-regulation. BS and microglia cultures per se did notshow statistically significant responses to IL6, IL10, and ILlb at themRNA level, however, μBS were able to elicit a response which otherwisewas not induced in BS after LPS treatment. This indicates that theinteraction between microglia and CNS cells (such as neurons, astrocytesand oligodendrocytes) are required for a specific inflammatory response.This study demonstrates the importance of including microglialpopulations in in vitro models to better predict human pathogenicity.

Flavivirus (DENY-1, ZIKV-BR, and ZIKV-UG) infection induced differentresponses depending of the model used (microglia, BS or μBS). Flavivirusinfection resulted in produced a higher number of RNA virus copies inmicroglia cells (FIG. 12A). Moreover, these viruses were also able toreplicate in BS, which is rarely observed in vitro. There was an initialincrease in viral replication when microglia were added to BS (FIG.12A), however, levels of RNA copies/mL were similar between BS and μBSat 72 h. Microglia showed no changes in viability after virus infection(FIG. 12C). ZIKV-BR infection led to higher replication compared toZIKV-UG and DENY-1 independent of the model, which could indicate thatthis flavivirus strain is more aggressive than the others (FIG. 12A).Moreover, ZIKV-BR infection showed a higher replication rate inmicroglia and μBS than in BS (FIG. 12A) indicating that microglia maysomehow influence the replication rate.

Flavivirus infection in μBS and BS showed differences in the expressionprofile of the genes studied. After infection of μBS by ZIKV-BR andZIKV-UG, upregulation of TNF-α, IL6, IL1b, and CCL2 was observed whencompared with BS (FIG. 12D). Moreover, DENY-1 infection led to theupregulation of CCL2 and IL-1β in the presence of microglia (OS) andupregulation of TNF-α and IL-6 in microglia alone. The μBS modelpresented a stronger upregulation of all cytokines studied after ZIKVinfection, indicating that the interaction between BS and microgliaproduce more drastic changes in gene expression, which is more relevantto study the physiological response to infection. Moreover, ZIKV-BRinfection led to G2/M arrest in μBS when compared to ZIKV-UG and DENY-1(FIG. 12B). Similar results have demonstrated that ZIKVBR infectionderegulates the cell cycle, resulting in attenuated hNPC growth (Dang etal., 2016; Garcez et al., 2016; Tang et al., 2016; Ghouzzi et al.,2017). Furthermore, phagocytosis of apoptotic cells by microglia cellshas been described as a general mechanism to clear these cells fromtissues (Arandjelovic and Ravichandran, 2015). Duque and Descoteauxdescribed that beside the secretion of cytokines, macrophages canincrease their phagocytotic process, contributing to the defense againstpathogens (Arango Duque and Descoteaux, 2014).

The findings indicate that the microglia-driven inflammatory response toZIKV infection may be exacerbated by endogenous signaling molecules suchas viral proteins that can contribute to the pathological damage ofneurons. Based on these findings, the activated microglia in μBS play apotential role in enhancing the expression of pro-inflammatory cytokinessuch as interleukin (IL)-1β, IL-6, and TNF-α in response to ZIKV-BRinfection.

In conclusion, the incorporation of human microglia in BS produced aninflammatory response following LPS or flavivirus exposure. This has notbeen studied previously in a 3D in vitro brain model which includes apopulation of microglial cells. Both exposures produced changes in cellcycle and Annexin V analysis in presence of microglia, indicating thatthe microglia-driven inflammatory response induces cytotoxicity leadingto a decrease in cell viability. Without being bound by theory,microglia derived from iPSC-derived microglia can represent animprovement of this model to limit proliferation and extend theco-culture period. As microglia occupy a central position in the defenseand maintenance of the CNS, a 3D in vitro model including microglia canbe fundamental for studies of the brain. Moreover, variousanti-inflammatory drugs have been identified in treatingmicroglia-mediated neuroinflammation in the CNS (Baby et al., 2014).Without wishing to be bound by theory, the disclosed model can serve asa potential tool for the screening of therapeutic targets inneurological disorders and recovery from brain injury. In conclusion,the findings indicate that the μBS model has potential applications as aphysiologically relevant model to study infectious disease,host-pathogen interactions, and neuro-inflammation.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. An in vitro brain microphysiological system(BMPS), comprising at least one neural cell type aggregated into aspheroid mass; and a population of microglia-like cells, wherein the invitro BMPS is electrophysiologically active in a spontaneous manner. 2.The BMPS of claim 1, wherein the micro-glia like cells comprisemicroglia, microglia precursor cells, or a combination thereof.
 3. TheBMPS of claim 1, wherein the micro-glia like cells comprise monocytes,human monocytes, pro-monocyte cell lines, hematopoietic stem cells,isolated microglia, immortalized microglia, or combinations thereof. 4.The BMPS of claim 3, where the monocytes comprise adult cell-derivedmonocytes, embryonic cell-derived monocytes, or a combination thereof.5. The BMPS of claim 4, wherein the monocytes comprise embryonic stemcell (ESC)-derived monocytes, induced pluripotent stem cell(iPSC)-derived monocytes, or a combination thereof.
 6. The BMPS of claim3, wherein the isolated microglia comprise adult microglia, fetalmicroglia, or a combination thereof.
 7. The BMPS of claim 3, wherein themicroglia-like cells are derived from somatic cells, neuronal cells,myeloid progenitor cells, or a combination thereof.
 8. The BMPS of claim1, wherein the microglia-like cells express one or more of the followingbiomarkers: HLA-DR, Iba1, CD14, CX3CR1, F4/80, CD80, CD86, CD36, iNOS,COX2, ARG1, PPARγ, SOCS-3, TMEM119, Mertk, Ax1, CD11b, CD11c, P2RY12,CD45, CD68, CD40, B7, ICAM-1, or any combination thereof.
 9. The BMPS ofclaim 1, wherein the BMPS expresses receptors associated with microgliafunction.
 10. The BMPS of claim 9, wherein the receptors associated withmicroglia function comprise CCL2, CX3CL, RAGE, NLRP3, SR-AI, TREM2,FPRL1/FPR2, CD36, CD33, C5a, CR1, CR3/Mac-1, FcRs, FPRs, TLRs, or acombination thereof.
 11. The BMPS of claim 1, the BMPS being configuredto elicit a pro-inflammatory response, an anti-inflammatory response, ora combination thereof.
 12. The BMPS of claim 11, the BMPS beingconfigured to elicit a pro-inflammatory response to viral infection, LPSexposure, or a combination thereof.
 13. The BMPS of claim 11, the BMPSbeing configured to elicit an anti-inflammatory response to IL-3, IL-4,IL-10, IL-13, IL-1β, IL-6, TNF-α, TGF-β, or a combination thereof. 14.The BMPS of claim 1, wherein the microglia-like cells comprise about 20%or less of the BMPS.
 15. The BMPS of claim 1, wherein the at least oneneuronal cell type comprises a mature neuron, a glial cell, or acombination thereof.
 16. The BMPS of claim 15, wherein the at least oneneural cell type further comprises astrocytes, polydendrocytes,oligodendrocytes, or combinations thereof.
 17. The BMPS of claim 1,wherein the in vitro BMPS has neural characteristics selected from thegroup consisting of synaptogenesis, neuron-neuron interactions,neuronal-glial interactions, axon myelination, cell migration,neurological development, disease phenotypes, and combinations thereof.18. The BMPS of claim 17, wherein disease process phenotypes compriseautophagy, integrated stress response, non-sense mediated decay,lesions, amyloid deposition, plaque formation, protein aggregation, orcombinations thereof.
 19. The BMPS of claim 1, wherein the at least oneneural cell type express one or more biomarker selected from the groupconsisting of MBP, PLP, NG2, Olig1, Olig2, Olig 3, OSP, MOG, SOX10,neurofilament 200 (NF200), GRIN1, GAD1, GABA, TH, LMX1A, FOXO1, FOXA2,FOXO4, CNP, TH, TUBIII, NEUN, SLC1A6, and any combination thereof. 20.The BMPS of claim 1, wherein the at least one neuronal cell typecomprises one or more genetically modified cells.
 21. The BMPS of claim20, wherein the one or more genetically modified cells comprise one ormore reporter genes.
 22. The in vitro BMPS of claim 1, wherein thespheroid mass comprises a diameter that is about 1000 μm or less. 23.The in vitro BMPS of claim 22, wherein the spheroid mass comprises adiameter that is about 500 μm or less.
 24. The BMPS of claim 1, furthercomprising one or more endothelial cells, pericytes, or a combinationthereof capable of forming a blood-brain-barrier.
 25. A method ofreproducibly producing an in vitro brain microphysiological system(BMPS), comprising: inducing one or more pluripotent stem cell (PSC)types; differentiating the one or more PSC types to form one or moreneural progenitor cell (NPC) types; exposing the one or more NPC typesto gyratory shaking or stirring; differentiating the one or more NPCtypes into one or more neural cell types aggregated into a spheroidmass; and adding microglia-like cells.
 26. The method of claim 25,wherein the micro-glia like cells comprise mature microglia, monocytes,human monocytes, pro-monocyte cell lines, PSC-derived monocytes,hematopoetic stem cells, isolated microglia, immortalized microglia, orcombinations thereof.
 27. The method of claim 25, wherein the one ormore pluripotent stem cells are selected from the group consisting ofhuman or animal embryonic stem cells, iPSC, adult stem cells,fibroblasts, embryonic fibroblasts, peripheral blood mononuclear cells,neuronal precursor cells, mesenchymal stem cells, neuronal cells, glialcells, and combinations thereof.
 28. The method of claim 25, wherein themicroglia-like cells are added during an early stage (before BMPSdifferentiation) or late stage (after BMPS differentiation).
 29. Themethod of claim
 25. wherein microglia are generated in parallel with theBMPS.
 30. The method of claim 25, wherein gyratory shaking comprisesconstant or regular gyratory shaking or stirring for 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more weeks.